Werner G. Bergen

Intestinal Nitrogen Recycling and Utilization in Health and Disease

The role of intestinal microflora in digestive and metabolic processes has received increasing attention from researchers and clinicians. Both enterocytes and small intestine luminal microorganisms can degrade peptides and amino acids (AA). Further, enterocytes can utilize ammonia via glutamate, glutamine, citrulline, and urea synthesis, whereas luminal microbes will deaminate AA, hydrolyze luminal urea, and recycle this ammonia by synthesis of new microbial cells. Although, undoubtedly, some indispensable AA may arise from N cycling and microbial synthesis in the intestinal lumen, the actual net impact on protein nutrition status appears to be limited in humans and animals. Moreover, potential contributions of the recycled N as colonic luminal microbial proteins to AA in blood depend on colonic protein digestion and AA absorption. Finally, new evidence indicates that gut microbial metabolism may be enhanced by prebiotics and probiotics, with the prospects of new treatment paradigms for eliminating undesirable secondary N metabolites and ameliorating complications in whole-body N metabolism under the conditions of intestinal stress, liver disease, and kidney failure.

Lipid metabolism related gene-expression profiling in liver, skeletal muscle and adipose tissue in crossbred Duroc and Pietrain Pigs.

Body-weight differences in animals may be ascribed to genetic and environmental factors. Here we utilized two divergent porcine genotypes, the highly muscled, leaner PietrianxYorkshire pigs and less muscled, fatter DurocxYorkshire growing pigs (75-110 kg), to examine the role of genetic background on expression of genes associated with anabolic (Fatty acid synthase, FAS; glucose transporter 4, GLUT-4; stearoyl CoA desaturase, SCD; Sterol regulatory binding protein-1, SREBP-1; leptin) and catabolic lipid metabolism (Carnitine palmitoyltransferase-1B, CPT-1B; acyl-CoA dehydrogenase, ACDH) in adipose tissue (AT), liver (L) and skeletal muscle (SKM). Pietrain pigs had lower mRNA abundance for FAS, SREBP-1, SCD and leptin in AT and L, but higher mRNA abundance for L ACDH and SKM ACDH and CPT-1B than Durocs. Duroc pigs exhibited higher expression of FAS, SREBP-1, SCD, leptin in AT and FAS in L and lower expression of ACDH and CPT-1B in L SKM. GLUT-4 expression did not differ in SKM between the two genotypes. Feeding of a beta adrenergic agonist (Paylean) for 52 days lowered expression of lipid anabolic and enhanced lipid catabolic genes expressions similarly in both genotypes. Overall, the lipid metabolism genes differential expression patterns documented here showed that in Pietrain pigs mRNA abundances of synthesis genes were lower and of catabolic genes were higher than in Duroc pigs.

Emerging roles of zinc finger proteins in regulating adipogenesis

Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved in regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. Due to the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood in order to develop novel and long-term impact strategies for ameliorating obesity. In this review, we discuss recent work that has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken together, these data lead to the conclusion that ZFPs may become promising targets to combat human obesity.

The effect of ractopamine hydrochloride on gene expression in adipose tissues of finishing pigs.

The long-term effect of feeding the catecholamine analog ractopamine (RAC; ractopamine hydrochloride, Elanco Animal Health, Indianapolis, IN) on the expression of genes involved in energy and lipid metabolism in subcutaneous adipose tissue was studied. Large White pigs (84 kg) were fed corn- and soybean meal-based diets supplemented with 0, 20, or 60 mg/kg of RAC for 14, 28, or 42 d. Expression (mRNA abundance) in adipose tissue of sterol regulatory binding protein-1 (SREBP-1), PPARα, PPARγ2, fatty acid synthase (FAS), glucose transporter 4 (GLUT4), and stearoyl-CoA desaturase was determined by Northern blotting. Feed intakes did not differ, and RAC (20 and 60 mg/kg) improved BW gain at d 14, 28, and 42 (P < 0.05) and increased loin eye area (measured on d 42 only; P < 0.05). Expression of SREBP-1 and PPARγ2 declined (P < 0.05) with RAC by d 28 and 42, whereas expression of PPARα was increased (P < 0.05) on d 14, 28, and 42. After 14 d, expression of FAS and GLUT4 was decreased (P < 0.05) with 60 mg/kg of RAC, whereas both RAC concentrations attenuated FAS expression on d 28 and 42. Overall, adipose tissue stearoyl-CoA desaturase expression was not affected by RAC but showed somewhat less expression (P < 0.15) on d 28 at 60 mg/kg of RAC. Although prolonged, chronic RAC feeding most likely downregulates adipose tissue membrane β-adrenergic receptors, mRNA abundances of anabolic lipid metabolism transcription factors, glucose transporters, and enzymes (SREBP-1, PPARγ2, FAS, GLUT4) were still attenuated up to d 42. Conversely, a transcription factor related to oxidative metabolism expression (PPARα) was enhanced. We conclude that even after 42 d, RAC still decreased expression of lipogenic genes in adipose tissue by yet undefined cyclic adenosine monophosphate-directed mechanisms, but in contemporary lean pigs, this effect is likely of limited practical significance.

Obesity, Metabolic Syndrome, and Adipocytes

Obesity and metabolic syndromes are examples whereby excess energy consumption and energy flux disruptions are causative agents of increased fatness. Because other, as yet elucidated, cellular factors may be involved and because potential treatments of these metabolic problems involve systemic agents that are not adipose depot-specific in their actions, should we be thinking of adipose depot-specific (cellular) treatments for these problems? For sure, whether treating obesity or metabolic syndrome, the characteristics of all adipose depot-specific adipocytes and stromal vascular cells should be considered. The focus of this paper is to begin to align metabolic dysfunctions with specific characteristics of adipocytes.

Dedifferentiated adipocyte-derived progeny cells (DFAT cells): Potential stem cells of adipose tissue.

Analyses of mature adipocytes have shown that they possess a reprogramming ability in vitro, which is associated with dedifferentiation. The subsequent dedifferentiated fat cells (DFAT cells) are multipotent and can differentiate into adipocytes and other cell types as well. Mature adipocytes can be easily obtained by biopsy, and the cloned progeny cells are homogeneous in vitro. Therefore, DFAT cells (a new type of stem cell) may provide an excellent source of cells for tissue regeneration, engineering and disease treatment. The dedifferentiation of mature adipocytes, the multipotent capacity of DFAT cells and comparisons and contrasts with mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPS) are discussed in this review.

Adipose depots differ in cellularity, adipokines produced, gene expression, and cell systems

The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them—including stem cells—during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology.

Cell culture purity issues and DFAT cells

Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

Cell supermarket: adipose tissue as a source of stem cells.

Adipose tissue is derived from numerous sources, and in recent years this tissue has been shown to provide numerous cells from what seemingly was a population of homogeneous adipocytes. Considering the types of cells that adipose tissue-derived cells may form, these cells may be useful in a variety of clinical and scientific applications. The focus of this paper is to reflect on this area of research and to provide a list of potential (future) research areas.

Bovine dedifferentiated adipose tissue (DFAT) cells: DFAT cell isolation

Dedifferentiated fat cells (DFAT cells) are derived from lipid-containing (mature) adipocytes, which possess the ability to symmetrically or asymmetrically proliferate, replicate, and redifferentiate/transdifferentiate. Robust cell isolation and downstream culture methods are needed to isolate large numbers of DFAT cells from any (one) adipose depot in order to establish population dynamics and regulation of the cells within and across laboratories. In order to establish more consistent/repeatable methodology here we report on two different methods to establish viable DFAT cell cultures: both traditional cell culture flasks and non-traditional (flat) cell culture plates were used for ceiling culture establishment. Adipocytes (maternal cells of the DFAT cells) were easier to remove from flat culture plates than flasks and the flat plates also allowed cloning rings to be utilized for cell/cell population isolation. While additional aspects of usage of flat-bottomed cell culture plates may yet need to be optimized by definition of optimum bio-coating to enhance cell attachment, utilization of flat plate approaches will allow more efficient study of the dedifferentiation process or the DFAT progeny cells. To extend our preliminary observations, dedifferentiation of Wagyu intramuscular fat (IMF)-derived mature adipocytes and redifferentiation ability of DFAT cells utilizing the aforementioned isolation protocols were examined in traditional basal media/differentiation induction media (DMI) containing adipogenic inducement reagents. In the absence of treatment approximately 10% isolated Wagyu IMF-mature adipocytes dedifferentiated spontaneously and 70% DFAT cells displayed protracted adipogenesis 12 d after confluence in vitro. Lipid-free intracellular vesicles in the cytoplasm (vesicles possessing an intact membrane but with no any observable or stainable lipid inside) were observed during redifferentiation. One to 30% DFAT cells redifferentiated into lipid-assimilating adipocytes in the DMI media, with distinct lipid-droplets in the cytoplasm and with no observable lipid-free vesicles inside. Moreover, a high confluence level promoted the redifferentiation efficiency of DFAT cells. Wagyu IMF dedifferentiated DFAT cells exhibited unique adipogenesis modes in vitro, revealing a useful cell model for studying adipogenesis and lipid metabolism.