Standardized In Vitro Models of Human Adipose Tissue Reveal Metabolic Flexibility in Brown Adipocyte Thermogenesis.

We used an immunohistochemical method for the inner mitochondrial membrane uncoupling protein (UCP) to examine whether human brown adipose tissue UCP could be detected by an anti-rat UCP antibody. Samples of human brown adipose tissue were obtained at medicolegal autopsies. Fat tissue was excised from around the common carotid arteries and in the subscapular region and from around the thoracic aorta. The subjects were either known alcohol consumers, in which thermogenically active brown adipose tissue (BAT) is often found, or victims of sudden infant death syndrome (SID). UCP was detected in all the cases examined, even when the post-mortem time from death to autopsy reached several days, but the intensity of the immunostaining was variable. Intense staining was observed in three cases with a post-mortem time under 24 hr, but in the SID cases a strong positive staining was seen even with a post-mortem delay of 4 days. These results show that an anti-rat UCP antibody can be used for immunohistochemical detection of UCP in human brown adipose tissue and that it provides a useful method for distinguishing between white and brown fat in paraffin-embedded samples. It can be used to detect UCP in the BAT of obese and diabetic individuals and probably also in the histopathological diagnosis of brown adipose tissue lipoma, known as hibernoma.

Human brown adipose tissue (BAT) activity decreases with age and obesity. In addition to uncoupling protein 1 (UCP1), several genetic markers of BAT in humans have been published. However, the link between human BAT activity and genetic markers has been inadequately explored. White adipose tissue (WAT) and BAT biopsies were obtained from 16 patients undergoing deep neck surgery. In vitro differentiated adipocytes were used to measure norepinephrine-stimulated mitochondrial uncoupling as a measure of in vitro BAT activity. Gene expression was determined in adipose tissue biopsies. Norepinephrine increased in vitro BAT activity in adipocytes derived from human BAT, and this increase was abolished by propranolol. Furthermore, in vitro BAT activity showed a negative correlation to age and BMI. UCP1 messenger RNA (mRNA) expression showed a positive correlation to in vitro BAT activity, while zinc finger protein of cerebellum 1 (ZIC1) mRNA showed a negative correlation to in vitro BAT activity. In human BAT biopsies, UCP1 mRNA showed negative correlations to age and BMI, while ZIC1 mRNA showed positive correlations to age and BMI. Differentiated adipocytes derived from human BAT maintain intrinsic characteristics of the donor. High ZIC1 mRNA does not necessarily reflect high BAT activity.

Brown adipocytes consume higher amounts of metabolic substrates and regulators, including thiamine, during adrenergic stimulation, supporting heat generation. Our previous findings showed that fedratinib, a potent inhibitor of thiamine transporter (ThTr) 2 and Janus kinase 2 (JAK2), reduced thermogenic activity; however, the underlying molecular mechanisms remain elusive. Primary human subcutaneous (SC) and deep neck (DN)-derived adipocytes were treated with dibutyryl (db)-cAMP, fedratinib, or the combination of the two compounds after differentiation. Global transcriptomic analysis was performed by bulk RNA-sequencing. Differentially expressed genes were subjected to pathway enrichment analysis. We also utilized publicly available single-cell RNA-sequencing datasets and Adipose Tissue Knowledge Portal to correlate ThTr2 expression in adipose tissue to clinical parameters of patient cohorts. Amino acid flux was measured by metabolomics. ThTr2 expression was observed exclusively enriched in the adipocytes cluster within human brown and white adipose tissue. In response to ThTr2 inhibition, the db-cAMP-stimulated upregulation of the canonical thermogenic markers and proton leak respiration, which associates with UCP1-dependent heat generation, was prevented in both adipocyte types. RNA-sequencing found 40 and 41 downregulated genes potentially underlying the metabolic changes in SC and DN-derived adipocytes, respectively, which were involved in various biological pathways, including transcriptional regulation of brown and beige adipocytes differentiation, signaling by interleukins, nicotinamide salvaging, and gene and protein expression by JAK/STAT signaling after interleukin-12 stimulation. The expression of recently identified thermogenesis regulators, such as transglutaminase (TGM) 2 and inhibitor of DNA binding (ID) 1, was also abrogated by ThTr2 inhibition during adrenergic stimulation. Intriguingly, glutamate transporter (GLT) 1 and L-amino acid transporter (LAT) 2 expression was also attenuated by fedratinib, restricting amino acid consumption. Finally, we found that the expression of ThTr2 in human white adipose tissue was inversely correlated with body mass index, waist-hip ratio, leptin secretion, and plasma insulin, glucose, cholesterol, and triacylglycerol levels, supporting the importance of thiamine metabolism in adipocyte and metabolic health.

<p dir="ltr">Obesity is a growing global health concern, currently affecting over one billion individuals worldwide and significantly increasing the risk of cardiovascular, metabolic, and many other diseases. White adipose tissue (WAT), a highly heterogeneous and metabolically active organ, plays a central role in maintaining energy homeostasis and regulating the endocrine system. The distribution and cellular composition of WAT, which vary markedly among individuals and anatomical depots, are critical determinants of metabolic health. While recent single-cell technologies have advanced our understanding of WAT cytoarchitecture, current studies predominantly focus on subcutaneous and omental visceral fat, often overlooking the diversity within visceral depots. Furthermore, adipocytes present technical challenges for single-cell RNA sequencing due to their physical properties, prompting the use of single-nucleus RNA sequencing (snSeq), which introduces specific biases. Spatial transcriptomics (STx) offers an alternative approach, capturing transcriptomic information while preserving spatial context, albeit at a limited resolution. In this thesis, we integrated snSeq and STx data to investigate WAT heterogeneity in obesity. In <b>study I</b>, we identified novel adipocyte substates and associated one with insulin responsiveness. In <b>study II</b>, we systematically mapped WAT cell types across multiple datasets, linking cell-type composition to clinical traits. In <b>study III</b>, we examined spatial and cellular differences between intraperitoneal and subcutaneous depots, revealing depot-specific inflammatory signatures. Our findings underscore the significance of spatial and cellular context in adipose tissue, providing new insights into the molecular underpinnings of metabolic disease in obesity.</p><h3>List of scientific papers</h3><p dir="ltr">I. Backdahl J*, Franzen L*, Massier L, Li Q, <b>Jalkanen J,</b> Gao H, Andersson A, Bhalla N, Thorell A, Rydén M, Ståhl PL*, Mejhert N *. Spatial mapping reveals human adipocyte subpopulations with distinct sensitivities to insulin. Cell Metab. 2021 Sep 7;33(9):1869-1882.e6. <a href="https://doi.org/10.1016/j.cmet.2021.07.018" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.cmet.2021.07.018</a></p><p dir="ltr">II. Massier L, <b>Jalkanen J,</b> Elmastas M, Zhong J, Wang T, Nono Nankam PA, Frendo-Cumbo S, Bäckdahl J, Subramanian N, Sekine T, Kerr AG, Tseng BTP, Laurencikiene J, Buggert M, Lourda M, Kublickiene K, Bhalla N, Andersson A, Valsesia A, Astrup A, Blaak EE, Ståhl PL, Viguerie N, Langin D, Wolfrum C, Blüher M, Rydén M*, Mejhert N *. An integrated single cell and spatial transcriptomic map of human white adipose tissue. Nat Commun. 2023 Mar 15;14(1):1438. <a href="https://doi.org/10.1038/s41467-023-36983-2" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-023-36983-2</a></p><p dir="ltr">III. <b>Jalkanen J*</b>, Zhong J*, Nono Nankam PA, Bhalla N, Elmastas M, Luo J, Weinbrenner S, Frendo-Cumbo S, Pesti B, Gourash W, Courcoulas A, Yang Loureiro Z, Dietrich A, Bäckdahl J, Buggert M, Kalucka J, Emont MP, Rosen ED, Blüher M, Kovacs P, Ståhl PL, Massier L, Rydén M*, Mejhert N *. Cytoarchitectural Profiling of White Adipose Tissue Depots Links Serum Amyloid A Expressing Adipocytes to Immune Cell Activation. [Submitted] </p><p dir="ltr">* These authors contributed equally</p>

<p dir="ltr">Obesity is a growing global health concern, currently affecting over one billion individuals worldwide and significantly increasing the risk of cardiovascular, metabolic, and many other diseases. White adipose tissue (WAT), a highly heterogeneous and metabolically active organ, plays a central role in maintaining energy homeostasis and regulating the endocrine system. The distribution and cellular composition of WAT, which vary markedly among individuals and anatomical depots, are critical determinants of metabolic health. While recent single-cell technologies have advanced our understanding of WAT cytoarchitecture, current studies predominantly focus on subcutaneous and omental visceral fat, often overlooking the diversity within visceral depots. Furthermore, adipocytes present technical challenges for single-cell RNA sequencing due to their physical properties, prompting the use of single-nucleus RNA sequencing (snSeq), which introduces specific biases. Spatial transcriptomics (STx) offers an alternative approach, capturing transcriptomic information while preserving spatial context, albeit at a limited resolution. In this thesis, we integrated snSeq and STx data to investigate WAT heterogeneity in obesity. In <b>study I</b>, we identified novel adipocyte substates and associated one with insulin responsiveness. In <b>study II</b>, we systematically mapped WAT cell types across multiple datasets, linking cell-type composition to clinical traits. In <b>study III</b>, we examined spatial and cellular differences between intraperitoneal and subcutaneous depots, revealing depot-specific inflammatory signatures. Our findings underscore the significance of spatial and cellular context in adipose tissue, providing new insights into the molecular underpinnings of metabolic disease in obesity.</p><h3>List of scientific papers</h3><p dir="ltr">I. Backdahl J*, Franzen L*, Massier L, Li Q, <b>Jalkanen J,</b> Gao H, Andersson A, Bhalla N, Thorell A, Rydén M, Ståhl PL*, Mejhert N *. Spatial mapping reveals human adipocyte subpopulations with distinct sensitivities to insulin. Cell Metab. 2021 Sep 7;33(9):1869-1882.e6. <a href="https://doi.org/10.1016/j.cmet.2021.07.018" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.cmet.2021.07.018</a></p><p dir="ltr">II. Massier L, <b>Jalkanen J,</b> Elmastas M, Zhong J, Wang T, Nono Nankam PA, Frendo-Cumbo S, Bäckdahl J, Subramanian N, Sekine T, Kerr AG, Tseng BTP, Laurencikiene J, Buggert M, Lourda M, Kublickiene K, Bhalla N, Andersson A, Valsesia A, Astrup A, Blaak EE, Ståhl PL, Viguerie N, Langin D, Wolfrum C, Blüher M, Rydén M*, Mejhert N *. An integrated single cell and spatial transcriptomic map of human white adipose tissue. Nat Commun. 2023 Mar 15;14(1):1438. <a href="https://doi.org/10.1038/s41467-023-36983-2" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-023-36983-2</a></p><p dir="ltr">III. <b>Jalkanen J*</b>, Zhong J*, Nono Nankam PA, Bhalla N, Elmastas M, Luo J, Weinbrenner S, Frendo-Cumbo S, Pesti B, Gourash W, Courcoulas A, Yang Loureiro Z, Dietrich A, Bäckdahl J, Buggert M, Kalucka J, Emont MP, Rosen ED, Blüher M, Kovacs P, Ståhl PL, Massier L, Rydén M*, Mejhert N *. Cytoarchitectural Profiling of White Adipose Tissue Depots Links Serum Amyloid A Expressing Adipocytes to Immune Cell Activation. [Submitted] </p><p dir="ltr">* These authors contributed equally</p>

OBJECTIVEFibrosis is a newly appreciated hallmark of the pathological alteration of human white adipose tissue (WAT). We investigated the composition of subcutaneous (scWAT) and omental WAT (oWAT) fibrosis in obesity and its relationship with metabolic alterations and surgery-induced weight loss.RESEARCH DESIGN AND METHODSSurgical biopsies for scWAT and oWAT were obtained in 65 obese (BMI 48.2 ± 0.8 kg/m2) and 9 lean subjects (BMI 22.8 ± 0.7 kg/m2). Obese subjects who were candidates for bariatric surgery were clinically characterized before, 3, 6, and 12 months after surgery, including fat mass evaluation by dual energy X-ray absorptiometry. WAT fibrosis was quantified and characterized using quantitative PCR, microscopic observation, and immunohistochemistry.RESULTSFibrosis amount, distribution and collagen types (I, III, and VI) present distinct characteristics in lean and obese subjects and with WAT depots localization (subcutaneous or omental). Obese subjects had more total fibrosis in oWAT and had more pericellular fibrosis around adipocytes than lean subjects in both depots. Macrophages and mastocytes were highly represented in fibrotic bundles in oWAT, whereas scWAT was more frequently characterized by hypocellular fibrosis. The oWAT fibrosis negatively correlated with omental adipocyte diameters (R = −0.30, P = 0.02), and with triglyceride levels (R = −0.42, P < 0.01), and positively with apoA1 (R = 0.25, P = 0.05). Importantly, scWAT fibrosis correlated negatively with fat mass loss measured at the three time points after surgery.CONCLUSIONSOur data suggest differential clinical consequences of fibrosis in human WAT. In oWAT, fibrosis could contribute to limit adipocyte hypertrophy and is associated with a better lipid profile, whereas scWAT fibrosis may hamper fat mass loss induced by surgery.

Different Metabolic Responses of Human Brown Adipose Tissue to Activation by Cold and Insulin

Obesity-associated metabolic abnormalities comprise a cluster of conditions including dyslipidemia, insulin resistance, diabetes and cardiovascular diseases that has affected more than 650 million people all over the globe. Obesity results from the accumulation of white adipose tissues mainly due to the chronic imbalance of energy intake and energy expenditure. A variety of approaches to treat or prevent obesity, including lifestyle interventions, surgical weight loss procedures and pharmacological approaches to reduce energy intake and increase energy expenditure have failed to substantially decrease the prevalence of obesity. Brown adipose tissue (BAT), the primary source of thermogenesis in infants and small mammals may represent a promising therapeutic target to treat obesity by promoting energy expenditure through non-shivering thermogenesis mediated by mitochondrial uncoupling protein 1 (UCP1). Since the confirmation of functional BAT in adult humans by several groups, approximately a decade ago, and its association with a favorable metabolic phenotype, intense interest on the significance of BAT in adult human physiology and metabolic health has emerged within the scientific community to explore its therapeutic potential for the treatment of obesity and metabolic diseases. A substantially decreased BAT activity in individuals with obesity indicates a role for BAT in the setting of human obesity. On the other hand, BAT mass and its prevalence correlate with lower body mass index (BMI), decreased age and lower glucose levels, leading to a lower incidence of cardio-metabolic diseases. The increased cold exposure in adult humans with undetectable BAT was associated with decreased body fat mass and increased insulin sensitivity. A deeper understanding of the role of BAT in human metabolic health and its interrelationship with body fat distribution and deciphering proper strategies to increase energy expenditure, by either increasing functional BAT mass or inducing white adipose browning, holds the promise for possible therapeutic avenues for the treatment of obesity and associated metabolic disorders.

Identification of an optimal threshold for detecting human brown adipose tissue using receiver operating characteristic analysis of IDEAL MRI fat fraction maps

Human brown adipose tissue (BAT) has become an attractive target to combat the current epidemical spread of obesity and its associated co-morbidities. Currently, information on its functional role is primarily derived from rodent studies. Here, we present the first comparative proteotype analysis of primary human brown adipose tissue versus adjacent white adipose tissue, which reveals significant quantitative differences in protein abundances and in turn differential functional capabilities. The majority of the 318 proteins with increased abundance in BAT are associated with mitochondrial metabolism and confirm the increased oxidative capacity. In addition to uncoupling protein 1 (UCP1), the main functional effector for uncoupled respiration, we also detected the mitochondrial creatine kinases (CKMT1A/B, CKMT2), as effective modulators of ATP synthase coupled respiration, to be exclusively expressed in BAT. The abundant expression and utilization of both energy expenditure pathways in parallel highlights the complex functional involvement of BAT in human physiology.

The identification of functional brown adipose tissue in human adults has intensified interest in exploiting thermogenic energy expenditure for the purpose of weight management. However, food intake and energy expenditure are tightly regulated and it is generally accepted that variation in one component results in compensatory changes in the other. In the context of weight loss, additional biological adaptations occur in an attempt to further limit weight loss. In the present review, we discuss the relationship between increasing energy expenditure and body weight in humans, including the effects of cold exposure. The data raise the possibility that some processes, particularly those involved in thermogenesis, induce less compensatory food intake for a given magnitude of additional energy expenditure, a state we term the 'thermogenic disconnect'. Although cold exposure increases thermogenesis and can putatively be exploited to induce weight loss, there are multiple adaptive responses to cold, of which many actually reduce energy expenditure. In order to optimally exploit either cold itself or agents that mimic cold for thermogenic energy expenditure, these non-thermogenic cold responses must be considered. Finally, the relative contribution of brown adipose tissue vs other thermogenic processes in humans remains to be defined. However, overall the data suggest that activation of cold-induced thermogenic processes are promising targets for interventions to treat obesity and its secondary metabolic complications. (J Diabetes Invest, doi:10.1111/j.2040-1124.2011.00158.x, 2011).

In spite of the long history of evidence for brown adipose tissue (BAT) in adult humans (1), the evidence of its functionality was missing. The thinking started to change when metabolically active ...

Statin therapy has shown pleiotropic effects affecting both mitochondrial function and inflammatory status. However, few studies have investigated the concurrent effects of statin exposure on mitochondrial function and inflammatory status in human subcutaneous white adipose tissue. In a cross-sectional study, we investigated the effects of simvastatin on mitochondrial function and inflammatory status in subcutaneous white adipose tissue of 55 human participants: 38 patients (19 females/19 males) in primary prevention with simvastatin (> 40 mg/d, > 3 mo) and 17 controls (9 females/8 males) with elevated plasma cholesterol. The 2 groups were matched on age, body mass index, and maximal oxygen consumption. Anthropometrics and fasting biochemical characteristics were measured. Mitochondrial respiratory capacity was assessed in white adipose tissue by high-resolution respirometry. Subcutaneous white adipose tissue expression of the inflammatory markers IL-6, chemokine (C-C motif) ligand 2 (CCL2), CCL-5, tumor necrosis factor-α, IL-10, and IL-4 was analyzed by quantitative PCR. Simvastatin-treated patients showed lower plasma cholesterol (P < .0001), low-density lipoprotein (P < .0001), and triglyceride levels (P = .0116) than controls. Simvastatin-treated patients had a lower oxidative phosphorylation capacity of mitochondrial complex II (P = .0001 when normalized to wet weight, P < .0001 when normalized to citrate synthase activity [intrinsic]), and a lower intrinsic mitochondrial electron transport system capacity (P = .0004). Simvastatin-treated patients showed higher IL-6 expression than controls (P = .0202). Simvastatin treatment was linked to mitochondrial respiratory capacity in human subcutaneous white adipose tissue, but no clear link was found between statin exposure, respiratory changes, and inflammatory status of adipose tissue.

Obesity is endemic in many regions of the world and a forerunner of several serious and sometimes fatal diseases such as ischemic heart disease, stroke, kidney failure and neoplasia. Although we know its origin--it results when energy intake exceeds energy expenditure--at present, the only proven therapy is bariatric surgery. This is a major abdominal procedure that, for reasons that are largely unknown (it cannot be explained solely by a reduction in ventricular volume), significantly reduces energy intake, but because of cost and limited availability, it will most likely be reserved for only a small fraction of those who stand to gain from effective antiobesity treatment. Clearly, alternative ways to treat obesity are needed. Another way to combat excessive accumulation of white adipose tissue would be to increase energy expenditure. Rodents, hibernators and human infants all have a specialized tissue--brown adipose tissue (BAT)--with the unique capacity to regulate energy expenditure by a process called adaptive thermogenesis. This process depends on the expression of uncoupling protein-1 (UCP1), which is a unique marker for BAT. UCP1 is an inner mitochondrial membrane protein that short circuits the mitochondrial proton gradient, so that oxygen consumption is no longer coupled to adenosine triphosphate synthesis. As a consequence, heat is generated. Mice lacking ucp-1 are severely compromised in their ability to maintain normal body temperature when acutely exposed to cold and they are also prone to become obese. We have shown that, in mice, BAT protects against diet-induced obesity, insulin resistance and type 2 diabetes. This is based on prevention of excessive accumulation of triglyceride in non-adipose tissues such as muscle and liver. Ectopic triglyceride storage at these locations is associated with initiation of insulin resistance and, ultimately, development of type 2 diabetes.

Perivascular adipose tissue (PVAT) is a complex tissue that is increasingly recognized for its roles in vascular health and disease. The form and function of PVAT changes depending on its anatomical location. Understanding cellular composition can give great insight into function. We had previously successfully performed single nucleus RNA-sequencing (snRNAseq) on brown fats, the thoracic aortic PVAT and subscapular brown adipose tissue (BAT) from Dahl SS rats. However, application of the same nuclei isolation method to white adipose tissue (WAT) depots (perivascular and non-perivascular) from the same rat strain resulted in insufficient yield of nuclei (&lt; 200 nuclei/mg) and RNA for downstream single-nuclei analyses. Similar challenges were encountered when processing bovine WAT, which exhibits similar processing difficulties to those observed with human WAT. This study aimed to optimize and validate nuclei isolation protocols from human and mouse WAT in rat and bovine samples. This is important because it is WAT that surrounds vessels that govern total peripheral resistance. Protocols were evaluated based on a) the quantity of nuclei isolated, b) quality of nuclei determined by microscopic visualization, and c) total RNA content of the nuclei pellet. A protocol utilizing homogenization and wash buffers designed for nuclei isolation from frozen human WAT, as well as liquid nitrogen pulverization and Dounce homogenization of tissues, proved translatable to both rat and bovine WAT depots (rat retroperitoneal fat: 3100 nuclei/mg tissue; rat mesenteric perivascular adipose tissue: 2200 nuclei/mg tissue; bovine white fat; 1450 nuclei/mg tissue) with key modifications. We developed protocol modifications including lipid removal techniques and optimization of centrifugation speed, as well as the downstream applications and results of nuclei fixation and sequencing for all depots: rat WATs and BATs (both perivascular and non-perivascular) and bovine WAT. Collectively, we illustrate the importance of a robust nuclei isolation protocol that is generalizable across distinct adipose depot locations and species for the evaluation of single-nuclei transcriptomic profiles. This developed protocol should prove useful for snRNAseq of WAT from multiple species.