Umesh D. Wankhade

Advances in Adipose-Derived Stem Cells Isolation, Characterization, and Application in Regenerative Tissue Engineering

Obesity is a complex, multifactorial disease that has been extensively researched in recent times. Obesity is characterized by excess deposition of adipose tissue in response to surplus energy. Despite the negative connotations of adipose tissue (AT), it serves as a critical endocrine organ. Adipose tissue is a source of several adipokines and cytokines which have been deemed important for both normal metabolic function and disease formation. The discoveries of metabolically active brown AT in adult humans and adipose tissue derived stem cells (ADSC) have been key findings in the past decade with potential therapeutic implications. ADSCs represent an enticing pool of multipotent adult stem cells because of their noncontroversial nature, relative abundance, ease of isolation, and expandability. A decade and a half since the discovery of ADSCs, the scientific community is still working to uncover their therapeutic potential in a wide range of diseases. In this review, we provide an overview of the recent developments in the field of ADSCs and examine their potential use in transplantation and cell-based therapies for the regeneration of diseased organs and systems. We also hope to provide perspective on how to best utilize this readily available, powerful pool of stem cells in the future.

Enhanced offspring predisposition to steatohepatitis with maternal high-fat diet is associated with epigenetic and microbiome alterations

Objective Non-alcoholic fatty liver disease (NAFLD) is an important co-morbidity associated with obesity and a precursor to steatohepatitis. However, the contributions of gestational and early life influences on development of NAFLD and NASH remain poorly appreciated. Methods Two independent studies were performed to examine whether maternal over-nutrition via exposure to high fat diet (HFD) leads to exacerbated hepatic responses to post-natal HFD and methionine choline deficient (MCD) diets in the offspring. Offspring of both control diet- and HFD-fed dams were weaned onto control and HFD, creating four groups. Results When compared to their control diet-fed littermates, offspring of HF-dams weaned onto HFD gained greater body weight; had increased relative liver weight and showed hepatic steatosis and inflammation. Similarly, this group revealed significantly greater immune response and pro-fibrogenic gene expression via RNA-seq. In parallel, 7–8 week old offspring were challenged with either control or MCD diets for 3 weeks. Responses to MCD diets were also exacerbated due to maternal HFD as seen by gene expression of classical pro-fibrogenic genes. Quantitative genome-scale DNA methylation analysis of over 1 million CpGs showed persistent epigenetic changes in key genes in tissue development and metabolism (Fgf21, Ppargc1β) with maternal HFD and in cell adhesion and communication (VWF, Ephb2) in the combination of maternal HFD and offspring MCD diets. Maternal HFD also influenced gut microbiome profiles in offspring leading to a decrease in α-diversity. Linear regression analysis revealed association between serum ALT levels and Coprococcus, Coriobacteriacae, Helicobacterioceae and Allobaculum. Conclusion Our findings indicate that maternal HFD detrimentally alters epigenetic and gut microbiome pathways to favor development of fatty liver disease and its progressive sequelae.

Melanocortin 4 Receptor is a Transcriptional Target of Nescient Helix-Loop-Helix-2

Melanocortin 4 receptor (Mc4r/MC4R) is a G-Protein coupled receptor that is expressed in the hypothalamus and implicated in body weight control. Mutations in MC4R are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation underlying the melanocortin pathway are far from being fully understood. The transcription factor nescient helix-loop-helix 2 (Nhlh2) influences the melanocortin pathway through transcriptional regulation of prohormone convertase I, which influences the production of melanocortin peptides. In the present study, Nhlh2s role as a transcriptional regulator of Mc4r has been demonstrated. Nhlh2 knockout mice have reduced hypothalamic expression of Mc4r mRNA, suggesting that it could be a direct or indirect transcriptional regulator of the Mc4r promoter. To demonstrate direct transcriptional regulation, chromatin immunoprecipitation and electrophoretic gel shift assays show that Nhlh2 binds to the E-Boxes located at -551, -366 and +54 on the Mc4r promoter. Leptin-induced transactivation of the Mc4r promoter is significantly higher in the presence of exogenously added Nhlh2. siRNA knockdown of Nhlh2 leads to significantly reduced endogenous Mc4r mRNA expression levels in N29/2 cell line. Transactivation using promoters with mutations in each of the E-Boxes results in significantly reduced transactivation efficiency compared to the WT Mc4r promoter, suggesting that Nhlh2 regulates Mc4r transcription through these sites. Findings from these studies, combined with previous work implicating Nhlh2 as a transcriptional regulator of both the Mc4r gene and the melanocortin pathway, suggest that Nhlh2s transcriptional activity directly influences the human and rodent body weight control pathways.

Novel Browning Agents, Mechanisms, and Therapeutic Potentials of Brown Adipose Tissue

Nonshivering thermogenesis is the process of biological heat production in mammals and is primarily mediated by brown adipose tissue (BAT). Through ubiquitous expression of uncoupling protein 1 (Ucp1) on the mitochondrial inner membrane, BAT displays uncoupling of fuel combustion and ATP production in order to dissipate energy as heat. Because of its crucial role in regulating energy homeostasis, ongoing exploration of BAT has emphasized its therapeutic potential in addressing the global epidemics of obesity and diabetes. The recent appreciation that adult humans possess functional BAT strengthens this prospect. Furthermore, it has been identified that there are both classical brown adipocytes residing in dedicated BAT depots and “beige” adipocytes residing in white adipose tissue depots that can acquire BAT-like characteristics in response to environmental cues. This review aims to provide a brief overview of BAT research and summarize recent findings concerning the physiological, cellular, and developmental characteristics of brown adipocytes. In addition, some key genetic, molecular, and pharmacologic targets of BAT/Beige cells that have been reported to have therapeutic potential to combat obesity will be discussed.

Deletion of Nhlh2 results in a defective torpor response and reduced beta adrenergic receptor expression in adipose tissue.

Background Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2), display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO) mouse a useful model to study the role of central nervous system (CNS) control on peripheral tissue such as adipose tissue. Methodology Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes. Principal Findings We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for β3-adrenergic receptors (β3-AR), β2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice. Conclusions These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals.

Transforming growth factor-β3 (TGF-β3) knock-in ameliorates inflammation due to TGF-β1 deficiency while promoting glucose tolerance.

Background: The mammalian TGF-β homologues have highly conserved heterologous motifs that are believed to impart isoform-specific functions. Results: Exchange of TGF-β1 ligand for TGF-β3 in a mouse reveals functional redundancies/differences in cell signaling. Conclusion: TGF-β1 and TGF-β3 are only partially interchangeable. Significance: These findings have relevance for therapeutic approaches aimed at targeting the TGF-β pathway in disease conditions. Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1Lβ3/Lβ3). In the TGF-β1Lβ3/Lβ3 mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1−/− mice, the TGF-β1Lβ3/Lβ3 mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1Lβ3/Lβ3 mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1Lβ3/Lβ3 mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease.

Persistent influence of maternal obesity on offspring health: Mechanisms from animal models and clinical studies

The consequences of excessive maternal weight and adiposity at conception for the offspring are now well recognized. Maternal obesity increases the risk of overweight and obesity even in children born with appropriate-for-gestational age (AGA) birth weights. Studies in animal models have employed both caloric excess and manipulation of macronutrients (especially high-fat) to mimic hypercaloric intake present in obesity. Findings from these studies show transmission of susceptibility to obesity, metabolic dysfunction, alterations in glucose homeostasis, hepatic steatosis, skeletal muscle metabolism and neuroendocrine changes in the offspring. This review summarizes the essential literature in this area in both experimental and clinical domains and focuses on the translatable aspects of these experimental studies. Moreover this review highlights emerging mechanisms broadly explaining maternal obesity-associated developmental programming. The roles of early developmental alterations and placental adaptations are also reviewed. Increasing evidence also points to changes in the epigenome and other emerging mechanisms such as alterations in the microbiome that may contribute to persistent changes in the offspring. Finally, we examine potential interventions that have been employed in clinical cohorts.

Gut microbiota are linked to increased susceptibility to hepatic steatosis in low-aerobic-capacity rats fed an acute high-fat diet

Poor aerobic fitness is linked to nonalcoholic fatty liver disease and increased all-cause mortality. We previously found that rats with a low capacity for running (LCR) that were fed an acute high-fat diet (HFD; 45% kcal from fat) for 3 days resulted in positive energy balance and increased hepatic steatosis compared with rats that were highly aerobically fit with a high capacity for running (HCR). Here, we tested the hypothesis that poor physiological outcomes in LCR rats following acute HFD feeding are associated with alterations in cecal microbiota. LCR rats exhibited greater body weight, feeding efficiency, 3 days of body weight change, and liver triglycerides after acute HFD feeding compared with HCR rats. Furthermore, compared with HCR rats, LCR rats exhibited reduced expression of intestinal tight junction proteins. Cecal bacterial 16S rDNA revealed that LCR rats had reduced cecal Proteobacteria compared with HCR rats. Microbiota of HCR rats consisted of greater relative abundance of Desulfovibrionaceae and unassigned genera within this family, suggesting increased reduction of endogenous mucins and proteins. Although feeding rats an acute HFD led to reduced Firmicutes in both strains, short-chain fatty acid-producing Phascolarctobacterium was reduced in LCR rats. In addition, Ruminococcae and Ruminococcus were negatively correlated with energy intake in the LCR/HFD rats. Predicted metagenomic function suggested that LCR rats had a greater capacity to metabolize carbohydrate and energy compared with HCR rats. Overall, these data suggest that the populations and metabolic capacity of the microbiota in low-aerobically fit LCR rats may contribute to their susceptibility to acute HFD-induced hepatic steatosis and poor physiologic outcomes.

Dynamic assessment of microbial ecology (DAME): a web app for interactive analysis and visualization of microbial sequencing data

Summary: Dynamic assessment of microbial ecology (DAME) is a Shiny‐based web application for interactive analysis and visualization of microbial sequencing data. DAME provides researchers not familiar with R programming the ability to access the most current R functions utilized for ecology and gene sequencing data analyses. Currently, DAME supports group comparisons of several ecological estimates of &agr;‐diversity and &bgr;‐diversity, along with differential abundance analysis of individual taxa. Using the Shiny framework, the user has complete control of all aspects of the data analysis, including sample/experimental group selection and filtering, estimate selection, statistical methods and visualization parameters. Furthermore, graphical and tabular outputs are supported by R packages using D3.js and are fully interactive. Availability and implementation: DAME was implemented in R but can be modified by Hypertext Markup Language (HTML), Cascading Style Sheets (CSS), and JavaScript. It is freely available on the web at https://acnc‐shinyapps.shinyapps.io/DAME/. Local installation and source code are available through Github (https://github.com/bdpiccolo/ACNC‐DAME). Any system with R can launch DAME locally provided the shiny package is installed. Contact: bdpiccolo@uams.edu

Cecal versus fecal microbiota in Ossabaw swine and implications for obesity

The gut microbiome plays a critical role in the onset and progression of obesity and the metabolic syndrome. However, it is not well documented whether the cecal vs. the fecal microbiome is more relevant when assessing their contributions to these diseases. Here, we amplified the V4 region of the 16S rRNA gene from cecal and fecal samples of female Ossabaw swine fed a low-fat control diet (10.5% fat, n = 4) or Western diet (43.0% fat, 17.8% high fructose corn syrup, 2% cholesterol; n = 3) for 36 wk. Obesity significantly lowered alpha-diversity ( P < 0.05), and there was clear separation in beta-diversity between lean and obese pigs, as well as between cecal and fecal samples ( P < 0.05). Obesity dramatically increased ( P < 0.05) the Firmicutes:Bacteroidetes ratio in fecal samples, and Actinobacteria was higher ( P < 0.05) in fecal vs. cecal samples in obese pigs. Cyanobacteria, Proteobacteria, and Fusobacteria were increased ( P < 0.05), while Spirochaetes, Tenericutes, and Verrucomicrobia were decreased ( P < 0.05) in obese vs. lean pigs. Prevotellaceae was reduced ( P < 0.05) in obese fecal vs. cecal samples. Moreover, cecal samples in obese had greater ( P < 0.05) predicted metabolic capacity for glycan biosynthesis and metabolism and LPS biosynthesis compared with fecal. Obese pigs also had greater ( P < 0.05) capacity for carbohydrate metabolism, which was driven by obese fecal rather than cecal samples and was opposite in lean pigs ( P < 0.05). The observed differences in pro-inflammatory microbiota and their metabolic capacity in cecal vs. fecal samples of obese pigs provide new insight into evaluating the microbiome in the pathogenesis of obesity and metabolic disease.