Youn-Kyoung Lee

β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolic genes in skeletal muscle and contributes to the response of muscle to exercise. Muscle PGC-1α transgenic expression and exercise both increase the expression of thermogenic genes within white adipose. How the PGC-1α-mediated response to exercise in muscle conveys signals to other tissues remains incompletely defined. We employed a metabolomic approach to examine metabolites secreted from myocytes with forced expression of PGC-1α, and identified β-aminoisobutyric acid (BAIBA) as a small molecule myokine. BAIBA increases the expression of brown adipocyte-specific genes in white adipocytes and β-oxidation in hepatocytes both in vitro and in vivo through a PPARα-mediated mechanism, induces a brown adipose-like phenotype in human pluripotent stem cells, and improves glucose homeostasis in mice. In humans, plasma BAIBA concentrations are increased with exercise and inversely associated with metabolic risk factors. BAIBA may thus contribute to exercise-induced protection from metabolic diseases.

Programming human pluripotent stem cells into white and brown adipocytes

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%–90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.

White-to-brown metabolic conversion of human adipocytes by JAK inhibition

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK–STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

Genetic Insulin Resistance is a Potent Regulator of Gene Expression and Proliferation in Human iPS Cells

Insulin resistance is central to diabetes and metabolic syndrome. To define the consequences of genetic insulin resistance distinct from those secondary to cellular differentiation or in vivo regulation, we generated induced pluripotent stem cells (iPSCs) from individuals with insulin receptor mutations and age-appropriate control subjects and studied insulin signaling and gene expression compared with the fibroblasts from which they were derived. iPSCs from patients with genetic insulin resistance exhibited altered insulin signaling, paralleling that seen in the original fibroblasts. Insulin-stimulated expression of immediate early genes and proliferation were also potently reduced in insulin resistant iPSCs. Global gene expression analysis revealed marked differences in both insulin-resistant iPSCs and corresponding fibroblasts compared with control iPSCs and fibroblasts. Patterns of gene expression in patients with genetic insulin resistance were particularly distinct in the two cell types, indicating dependence on not only receptor activity but also the cellular context of the mutant insulin receptor. Thus, iPSCs provide a novel approach to define effects of genetically determined insulin resistance. This study demonstrates that effects of insulin resistance on gene expression are modified by cellular context and differentiation state. Moreover, altered insulin receptor signaling and insulin resistance can modify proliferation and function of pluripotent stem cell populations.

White to brite adipocyte transition and back again

Identification and characterization of a third type of adipocyte known as brite (brown-in-white) adipocytes has drawn considerable attention, as these cells are thought to regulate energy expenditure and may help combat obesity. Remarkably, white adipocytes can adopt the characteristics of brite adipocytes following cold stimulation, and this process is reversible in vivo.

Efficient culturing and genetic manipulation of human pluripotent stem cells.

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.

Severe insulin resistance alters metabolism in mesenchymal progenitor cells.

Donohue syndrome (DS) is characterized by severe insulin resistance due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in DS in the undifferentiated state, we generated mesenchymal progenitor cells (MPCs) from induced pluripotent stem cells derived from a 4-week-old female with DS and a healthy newborn male (control). INSR mRNA and protein were significantly reduced in DS MPC (for β-subunit, 64% and 89% reduction, respectively, P < .05), but IGF1R mRNA and protein did not differ vs control. Insulin-stimulated phosphorylation of INSR or the downstream substrates insulin receptor substrate 1 and protein kinase B did not differ, but ERK phosphorylation tended to be reduced in DS (32% decrease, P = .07). By contrast, IGF-1 and insulin-stimulated insulin-like growth factor 1 (IGF-1) receptor phosphorylation were increased in DS (IGF-1, 8.5- vs 4.5-fold increase; INS, 11- vs 6-fold; P < .05). DS MPC tended to have higher oxygen consumption in both the basal state (87% higher, P =.09) and in response to the uncoupler carbonyl cyanide-p-triflouromethoxyphenylhydrazone (2-fold increase, P =.06). Although mitochondrial DNA or mass did not differ, oxidative phosphorylation protein complexes III and V were increased in DS (by 37% and 6%, respectively; P < .05). Extracellular acidification also tended to increase in DS (91% increase, P = .07), with parallel significant increases in lactate secretion (34% higher at 4 h, P < .05). In summary, DS MPC maintain signaling downstream of the INSR, suggesting that IGF-1R signaling may partly compensate for INSR mutations. However, alterations in receptor expression and pathway-specific defects in insulin signaling, even in undifferentiated cells, can alter cellular oxidative metabolism, potentially via transcriptional mechanisms.

Differentiation of white and brown adipocytes from human pluripotent stem cells.

Given the rapid increase in the prevalence of obesity and related metabolic diseases, research to better understand adipose tissue biology and physiology has garnered considerable attention. Adipose has been studied using both cell culture systems and model organisms. However, the mechanisms of adipocyte regulation are not comprehensively understood, as currently available in vitro or in vivo systems do not fully recapitulate human metabolic processes. Human primary adipocytes are difficult to culture and expand, and current cell systems have limitations such as cell line-to-cell line variability for adipocyte differentiation, decreased proliferation, and differentiation potential upon continued passaging. To overcome these limitations, we developed and established an efficient and robust adipocyte differentiation protocol using human pluripotent stem cells (hPSCs) and inducible expression of key adipogenic transcriptional regulators. Here, we provide a simple and stepwise protocol for programming hPSCs into mature white or brown adipocytes.

Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease

Summary The striking rise of obesity-related metabolic disorders has focused attention on adipocytes as critical mediators of disease phenotypes. To better understand the role played by excess adipose in metabolic dysfunction it is crucial to decipher the transcriptional underpinnings of the low-grade adipose inflammation characteristic of diseases such as type 2 diabetes. Through employing a comparative transcriptomics approach, we identified IRF1 as differentially regulated between primary and in vitro-derived genetically matched adipocytes. This suggests a role as a mediator of adipocyte inflammatory phenotypes, similar to its function in other tissues. Utilizing adipose-derived mesenchymal progenitors we subsequently demonstrated that expression of IRF1 in adipocytes indeed contributes to upregulation of inflammatory processes, both in vitro and in vivo. This highlights IRF1s relevance to obesity-related inflammation and the resultant metabolic dysregulation.