Similarl to the glucose consumption, lactate production in GM increased throughout culture In the third and fourth week lactate production increased again. Metabolite turnover, analysed in conditioned media over the period of 4 weeks. Glucose uptake and lactate production in a human adipose-derived stem cells hASC cultured in growth media GM and b standard differentiation media DM.
In the present study, we investigated the outcome of high glucose and insulin application for fat cell augmentation in vitro , as published protocols for adipogenic differentiation vary among laboratories.
Since mature adipocytes represent both numerically and functionally the principal element of fat tissue [ 14 ], we intended to create an in vitro monolayer model for the successive approximation to the physiological context of human adipose tissue.
Unlike previous studies, we were specifically interested in adipogenesis stimulating processes not in the differentiation induction period but in the late phase of ASC differentiation.
In order to evaluate the degree of differentiation and lipid accumulation, we quantified Oil-Red-O stained area and used qRT-PCR to assess whether morphological changes in terminally differentiated cells were still regulated by molecular processes. Isolated cells were confirmed as mesenchymal stem cells by the typical surface marker expression. As the SVF of adipose tissue is composed of a heterogeneous cell population, variable expression of hematopoietic and endothelial surface markers is commonly found at early passages [ 15 ].
Especially, literature differ respectively the expression of CD Although the International Society for Cellular Therapy ISCT declared CD34 as a negative marker in [ 10 ], in recent years, its expression has been discussed controversially [ 16 — 18 ].
Based on the great variability in data regarding the positivity of CD34, the ISCT re-evaluated the minimal criteria for stromal cells and admit a tissue, harvest and culture dependent expression of CD34, with a consistent downregulation according to the time of cultivation [ 19 ]. In the last years, numerous in vivo studies have elucidated the role of hyperinsulinemia in promoting adipogenesis, being associated with obesity and insulin resistance, resulting in severe metabolic dysfunctionality [ 20 ].
While insulin is known to stimulate adipocyte differentiation dose-dependently at the early stage of differentiation [ 21 ], its role in the late phase of differentiation is yet not completely understood. In this study, the capacity to accumulate intracellular lipids was assessed in the presence or absence of glucose and insulin 28 days after differentiation was induced. High insulin treatment of adipocytes was found to be associated with a significant reduction of intracytoplasmic lipid accumulation and a formation of multiple small vacuoles with only a few unilocular large ones, suggesting a more immature stage of conversion.
There is a lack of data for human cell lines; however, results are consistent with previous data obtained in 3T3-L1 adipocytes, describing long-term exposure to insulin eliciting a more immature phenotype with fewer unilocular lipid droplets [ 22 ]. Though we found these phenotypical observations accompanied by only marginal transcriptional changes of adipocyte-specific genes.
Nevertheless, it is not possible to exclude posttranscriptional changes in enzyme activity and synthesis rate that were not monitored in this study. Raynolds et al. High-glucose treatment increased lipid accumulation in a dose-dependent manner, accompanied by a rise in gene expression of adipogenesis regulating genes. Aguiari et al. Nevertheless, we still found glucose and insulin having an influence on gene expression in late adipogenic differentiation.
To assess whether the observed effects of high-glucose treatment were attributable to its osmotic pressure, cells were incubated in equimolar mannitol media.
Although osmotic stress mediated by mannitol did not enhance intracytoplasmic lipid accumulation, it markedly increased expression levels of adipogenic genes. The above-described results for mannitol allow to assume that gene expression and morphological characteristics are not necessarily correlated. As far as we know, to date, no study has described that osmotic pressure per se has an adipogenic potential, triggering the expression of typical genes.
While the hormonal regulation of adipogenesis has been widely studied, present results may open the search for currently unknown signal transduction pathways regulating glucose-associated processes with osmotic stress as a mediator.
To investigate this further, we analysed glucose and lactate levels of conditioned media over a period of 4 weeks. Although not only glucose but also lactate metabolism is known to be regulated by glucose and insulin concentrations [ 28 ], we observed no differences between treatment groups.
Interestingly, cells with higher lipid droplet formation did not show higher glucose consumption. Even though glucose-treated cells showed a statistically significant increase in lipid accumulation compared to cells in high insulin media, glucose uptake remained at the same level. In contrast, Crandall et al. Furthermore, they illustrated that large fat cells convert significantly more of the utilized glucose to lactate than smaller ones.
Interestingly, they found higher lactate release correlated to a reduced glucose conversion to triglycerides and CO 2. This flexibility of lactate production in relation to other products of glucose metabolism indicates that storage of triglyceride may not only be achieved by a rise in glucose uptake but also by a modified contribution of products released from glucose metabolism.
To date, there is no appropriate method applying an extracorporal volume augmentation of prior-harvested autologous fat tissue for the reconstruction of soft tissue defects. In this concern, the subcutaneous fat tissue depot with its accessibility and abundant stem cell reservoir provides a unique source for autologous tissue grafting.
By establishing a protocol for the late phase of adipogenesis, our data support the notion that glucose enhances lipid accumulation and gene expression of adipogenic markers, whereas the role of insulin, showing opposed effects, has not been described before. We were able to show that glucose and insulin which were thought to act synergistically have different effects on late adipogenic differentiation; however, there is still a need for clarification.
Unexpectedly, we demonstrated a novel aspect of mannitol that equally showed to increase expression levels of typical markers. We thank the Centre for Physiology of the Hannover Medical School for their advice and technical support. National Center for Biotechnology Information , U. Journal List Adipocyte v. Published online Jul 6. Kuhbier , Peter M.
Peter M. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC. Associated Data Data Availability Statement The authors confirm that the data supporting the findings of this study are available within the article. Introduction Soft tissue engineering is a growing field that addresses the clinical challenges associated with the structural and functional therapy of defects caused by congenital deformities, post-operative or post-traumatic loss of soft tissue.
Table 1. Open in a separate window. Table 2. Figure 1 , Table 3 Table 3. Figure 1. Oil-Red-O staining For the imaging of lipid accumulation, cells were stimulated in six-well culture plates with each medium for 28 days and stained with Oil-Red-O Serva, Heidelberg Germany as described previously [ 11 ]. Table 4. Results Cells exhibit ASC-typical phenotype characteristics In culture, hASC adhered to plastic, showed a uniform spindle-like shape and proliferated to confluence within an average time of two weeks.
Glucose and insulin have no effect on viability High glucose and insulin concentration did not show any effect on cell viability compared to standard differentiation protocol. Figure 2. Effect of glucose and insulin on lipid accumulation After differentiation was induced, ASC lost their fibroblastic morphology Figure 3 a and adopted a more spherical shape with multiple small lipid droplets which coalesced gradually during culture Figure 3 b—e. Figure 3.
Figure 4. Figure 5. Changes in gene expression To assess whether the observed differences were accompanied by changes in gene expression, the expression of adipogenic markers was analysed by qRT-PCR. Figure 6. Glucose and lactate metabolism To analyse the glucose and lactate metabolism, we measured the metabolic parameters in the conditioned media. Figure 7. Discussion In the present study, we investigated the outcome of high glucose and insulin application for fat cell augmentation in vitro , as published protocols for adipogenic differentiation vary among laboratories.
Conclusions To date, there is no appropriate method applying an extracorporal volume augmentation of prior-harvested autologous fat tissue for the reconstruction of soft tissue defects.
In an elegant study, Spalding et al. However, the turnover of adipose cells decreases in hypertrophic obesity [ 33 ]. The hypertrophic response involves pre-existing adipocytes that increase TG storage under insulin stimulation, achieving a two- to three-fold increase in their volume [ 34 ]. When there is a long-term exposure to a hypertrophic environment, adipocytes reduce the sensitivity to insulin, thus favoring AT dysfunction [ 35 ].
Since adipocytes cannot store further lipids under these conditions, new small adipocytes are formed from preadipocytes, especially in the VAT, and this may even be protective against metabolic impairment [ 36 ]. Indeed, several studies have observed that during caloric excess, the ASC compartment may be induced to proliferate and differentiate.
For instance, dietary inputs can modulate these two biological endpoints in adults [ 37 ]. Therefore, the ability of mature adipocytes to accumulate lipids and the ability of ASCs from the stromal vascular fraction SVF to form new adipocytes are the key processes underlying the regenerative capacity of AT.
Under physiological conditions, both white adipose tissue WAT and brown adipose tissue BAT are hypervascularized, and the adipose vasculature displays functional plasticity to comply with the metabolic demands of adipocytes. Moreover, blood vessels not only supply nutrients and oxygen to nourish adipocytes, but they also serve as a cellular reservoir to provide adipose precursor and stem cells that control the AT mass and function.
Thus, physiological AT remodeling also requires an adequate perfusion and subsequent delivery of nutrients into fat cells [ 41 ]. Indeed, insulin can affect the vascular endothelium of AT by inducing widespread vasodilatation and capillary recruitment without significant changes in the endothelial nitric oxide synthase eNOS in healthy individuals [ 42 , 43 ]. Furthermore, several studies have reported that insulin increased endothelial cell migration via activation of the PI3K-Akt-SREBPRac1 pathway [ 44 ], and enhanced new vessel formation via glycogen synthase kinase-beta3 and eNOS [ 45 ].
In addition to regulating the release and storage of lipids, AT functions as a large endocrine organ that regulates several aspects of whole-body physiology through the release of hormones, lipids and cytokines adipokines [ 46 ]. Research over the last two decades has shown that AT releases molecules such as cytokines and other proinflammatory molecules e.
However, AT can also secrete molecules that are associated with enhanced insulin sensitivity, such as adiponectin and the recently discovered branched fatty acid esters of hydroxy fatty acids FAHFAs [ 49 ]. An early study using omics approaches in mature 3T3-L1 adipocytes showed that the in vitro administration of insulin induced the expression or secretion of a total of 27 proteins mainly through post-translational modifications , including adipsin a serine protease that stimulates glucose transport for triglyceride accumulation , secreted acidic cysteine-rich protein SPARC involved in cell reorganization and angiogenesis , complement C3, collagen and other components of the extracellular matrix ECM [ 50 ].
Among the numerous adipokines with endocrine activity, including factors involved in fat storage and metabolism and eating behavior, some have been described to be regulated by insulin both in vivo and in vitro. Moreover, Halleux et al. In addition, insulin can regulate the WAT production of leptin, an adipokine known to control feeding behavior by activating anorexigenic neurons, as well as energy expenditure [ 56 ].
The rates of leptin biosynthesis are positively correlated with BMI and fat cell size, however, the expression of leptin is also affected by insulin, which chronically stimulates leptin storage by pre- and post-translational mechanisms [ 73 ], suggesting that the insulin-induced release of preformed leptin could contribute to circulating levels of this hormone.
Moreover, in vivo and in vitro studies have demonstrated that insulin increases leptin release by human subcutaneous abdominal and mammary AT, as well as in rat epididymal AT and 3T3-FA adipocytes [ 57 , 58 , 59 , 61 ]. Apelin is a newly identified fat-derived hormone which is strongly associated with obesity and hyperinsulinemia [ 74 ].
Insulin was found to enhance apelin expression, since in a diabetic mouse model, the lack of insulin production causes a large decrease in apelin expression in adipocytes [ 61 ]. The circulating and AT subcutaneous and visceral levels of chemerin were increased under hyperinsulinaemic conditions in women with polycystic ovary syndrome PCOS [ 63 ], as well as in ex vivo experiments on SAT and VAT explants of these subjects [ 63 ].
A potent and robust insulin-induced upregulation of lipocalin-2, a novel protein involved in obesity and diabetes, was also shown in VAT explants, occurring via the activation of both PI3K and MAPK signaling [ 64 ]. On the other hand, insulin appears to negatively regulate two adipokines: resistin and omentin. Resistin is a member of the newly discovered cysteine-rich secretory protein family and has been associated with reduced systemic insulin sensitivity [ 76 ].
The secretion of resistin was suppressed in a mouse model of hyperinsulinaemia and during 3T3-L1 adipocyte differentiation by the activation of proteins that induce the degradation of its transcript via PI3K, ERK or p38 mitogen-activated protein kinase MAPK independent pathways [ 65 , 66 , 67 , 77 ].
Recently however, omentin was shown to enhance insulin-mediated glucose transport with changes in basal glucose transport, indicating that it has no intrinsic insulin-mimetic activity but may increase insulin signaling via the IRS protein [ 80 ]. Vaspin visceral adipose tissue-derived serpin, serpinA12 is a member of the serine protease inhibitor family of serpins, whose expression shows the highest values when plasma insulin levels and obesity peak, while they are lower in the presence of diabetes [ 81 ].
Moreover, serum vaspin concentrations show diurnal fluctuations with a pre-prandial rise and a post-prandial fall, probably due to insulin level excursions, as also confirmed during an insulin tolerance test in healthy individuals with reduced vaspin serum concentrations shortly after insulin administration [ 69 , 70 ]. Altogether, these findings support the concept that nutritional status, and consequently insulin levels, directly affect the production and release of multiple adipokines, which in turn may regulate glucose homeostasis, insulin sensitivity and the energy balance.
Therefore, deciphering the molecular mechanism underlying insulin-induced adipokine release could potentially lead to new therapeutic interventions against obesity and diabetes.
IR is a condition reflecting the reduction of insulin-mediated glucose uptake into the key insulin-sensitive tissues and is usually characterized by high circulating insulin levels, due to the compensatory enhancement of pancreatic insulin secretion [ 82 ]. This could lead one to believe that AT expansion could be fostered by peripheral IR, since high insulin levels may induce glucose uptake, lipogenesis and lipolysis inhibition in this tissue. On the other hand, dysfunctional AT develops as a result of its expansion, and this may favor IR.
Lipotoxicity has emerged as a key factor underlying the development of metabolic abnormalities, both in the presence of dysfunctional AT, as well as with the partial or complete absence of AT, as observed in lipodystrophies. The reduced ability of the SAT to store TGs results in increased lipolysis and the ectopic accumulation of fatty acids as TGs in the pancreas, muscle and liver, as well as in VAT, leading to the typical metabolic alterations [ 84 ].
Moreover, AT is also responsible for generating ATP through fatty acid beta-oxidation in mitochondria, providing maintenance for a wide range of cellular processes, such as growth and differentiation [ 85 ].
The causative mechanisms mediating the development of IR in AT have been investigated less thoroughly than in the muscle and liver and appear to involve defects in multiple steps of insulin signaling downstream from the INSR [ 86 ].
Furthermore, long-term exposure to a high-energy diet can induce IR in AT by causing a reduction in INSR content, probably through the disruption of the lipid bi-layer of adipocytes due to activation of peroxidation processes [ 89 ]. Moreover, deficiency of leptin secretion or action in mice with obesity and lipodystrophy, respectively, can affect insulin signaling, leading to chronic hyperinsulinaemia associated with hyperglycemia due to a down-regulation of IRS-2, a key mediator of insulin signaling [ 91 ].
The expression levels of GLUT4 are reduced in adipose cells from insulin-resistant obese and prediabetic subjects i. GLUT4 protein levels in AT are also a marker of whole-body insulin sensitivity, measured with the euglycemic clamp technique [ 4 ]. Finally, a decreased relative abundance of fatty acid binding protein 4 FABP4 expression at the adipocyte plasma membrane has been reported in T2D obese subjects, and this may also contribute to the abnormalities in the storage of TGs [ 93 ].
All of these mechanisms i. Specifically, when glucose uptake and utilization by adipocytes are reduced, lipolysis will proceed unrestrained and circulating NEFA concentrations will increase [ 96 ], as in shown in the early days in obese nondiabetic subjects using 14 C-palmitate in combination with the insulin clamp technique [ 97 ]. AT is one of the first tissues to respond to a nutritional overload, with such alterations impacting other tissues. Interestingly, the severity of IR varies in relation to the different AT compartments, and visceral adiposity is strongly associated with whole-body IR, being responsible for a higher lipid load in the portal vein, compromising the hepatic glucose balance.
Increased adipocyte size is associated with higher serum insulin concentrations, IR and an increased risk of developing T2D [ 98 ]. Indeed, severely obese individuals with a healthy metabolic profile have smaller adipocytes and increased circulating adiponectin levels than obese individuals with adverse metabolic features [ 99 ]. On the other hand, reduced blood supply may represent another important factor for the impairment of in vivo insulin-mediated glucose uptake in AT [ 96 , ], and inappropriate blood supply to SAT and VAT, as observed during exaggerate adipocyte enlargement, may contribute to the reduction of in vivo insulin-mediated glucose uptake.
Indeed, vascular and endothelial dysfunction observed in obesity and insulin resistance largely results from the altered secretion of proinflammatory cytokines, the decreased release of adiponectin from AT and postprandial hyperglycemia [ ]. Moreover, insulin-mediated vasodilation may be differentially impaired in VAT compared to SAT in obese subjects, suggesting a role of altered vascular insulin signaling in promoting inappropriate rates of glucose uptake [ 96 ] and fat expansion [ ].
Insulin resistance in endothelial cells can potentially play a role in glucose homeostasis through at least two mechanisms: The lack of a vasodilator effect of insulin and the reduction of the transendothelial delivery of insulin to its target tissues.
Early studies in humans have shown that insulin-stimulated vasodilation could be a significant contributor to insulin-stimulated glucose uptake [ ] by a mechanism involving eNOS, which can be impaired in people with obesity or type 2 diabetes [ ].
Subsequent studies have suggested that an effect of insulin resulting in an increased number of perfused capillaries in a given tissue, a phenomenon known as capillary recruitment, may be more important for glucose tolerance than the insulin effect on total blood flow [ ]. Different studies in animal models of IR conducted using the tissue-specific gene-deletion of INSR have allowed for the elucidation of the physiological roles of insulin, as well as the mechanisms underlying the development of IR in specific tissues Table 2.
However, different results have been obtained according to the selectivity of INSR gene deletion e. Conversely, permanent abrogation of INSR expression in adipocytes AIRKO resulted in severe lipodystrophy, with metabolic abnormalities such as IR, altered glucose homeostasis, dyslipidemia and fatty liver disease, leading to decreased lifespan [ , ].
Furthermore, INSR is also critical in adipocyte survival, as observed in a murine model of inducible INSR inactivation in mature adipocytes that developed WAT and BAT loss, cold intolerance and metabolic syndrome, but was then able to restore AT dysfunction after 10—30 days through regeneration mechanisms [ ].
Recently, Merry et al. These data clarify the key role of INSR for AT development and function, highlighting its impact on the maintenance of glucose homeostasis and insulin sensitivity. Thus, the alterations of INSR expression in human AT could also be relevant for the development of metabolic complications in offspring if they occur at the time of pregnancy. Insulin and IGF-I play a synergistic role on several endpoints i.
As already reviewed, the human INSR gene maps on chromosome 19 and encodes two isoforms depending on the exclusion or inclusion of 12 amino acids in the C-terminal domain, respectively, by a post-transcriptional exon skipping process. The short isoform INSR-A is predominantly expressed in undifferentiated cells and contributes to prenatal development and tissue growth, whereas the expression of the long isoform INSR-B is enhanced in post-mitotic and differentiated cells and is largely responsible for the systemic metabolic action of insulin in adults [ ].
These events are also affected by growth factors, including insulin itself [ ]. Furthermore, both INSR isoforms are co-expressed in most cell types and can form homodimers i. However, the trafficking of INSR isoforms may be differentially regulated by specific ligands, and this could also affect downstream responses.
The resulting hybrid receptors HRs mediate different biological responses on the basis of ligand affinity and downstream signaling [ ].
However, the role of the distinct INSR isoforms in the development and function of human AT has not yet been fully clarified. The HRs were first identified in human placentae [ ], but are basically ubiquitous. HRs may play an important role in receptor signaling in normal and pathological tissues, particularly in human cancers.
Patients with T2D display an increased expression of HRs in AT compared to non-diabetic control patients [ ], and this could contribute to reduced insulin action on glucose uptake and the inhibition of pro-inflammatory responses, since the IGF-IR could act as a negative regulator of insulin signaling, as shown in preadipocytes [ ].
To date, information on the role of HRs in human AT and adipocyte differentiation is limited. Moreover, treatment of these preadipocytes with both insulin and IGFs resulted in high rates of proliferation and glucose accumulation, due to the partial agonism of insulin and IGF-I on their own and in cognate receptors [ ].
Recent experimental evidence suggests that the absence of one receptor cannot compensate for the lack of the other, suggesting the synergism of the two receptors. Despite IGF-IR deletion in vivo not being essential for the growth and development of BAT in the presence of the INSR, its expression is crucial for the full function of BAT in terms of cold acclimation and maintaining an appropriate balance of death and survival for fetal brown adipocytes Table 2 [ , ].
Another study reported that the adipocyte-specific deletion of INSR and IGF-IR led to inability to maintain body temperature, lipodystrophy, severe diabetes and ectopic fat deposition compared to IGF-IR loss alone, with a modest effect on fat physiology [ ].
The mechanisms underlying the effects of both INSR and IGF-IR deficiency on AT may have an epigenetic origin, as recently found in brown adipocyte precursors lacking both receptors, which show a drop in several maternally and paternally expressed imprinted genes and miRNAs in a stable and heritable manner [ ].
Over the last few decades, specific insulin analogs have been designed to improve metabolic outcomes in diabetic patients by minimizing glycemic excursions and the risk of hypoglycemia [ ]. Short-acting analogs can be administered shortly before a meal, since they rapidly disassemble in the subcutaneous injection site and are easily absorbed in blood capillaries.
In contrast, long-acting analogs are usually administered once daily and allow for a slow and continuous method of insulin delivery. AspB10 was the first insulin analog to be developed, showing an increased affinity for both the INSR and IGF-IR with a high carcinogenic risk, and it is thus not used in clinical practice [ ].
The selective effects of the various insulin analogs on the INSR isoform have been investigated by Sciacca et al. The level of INSR-A and INSR-B tyrosine phosphorylation after stimulation with short- or long-acting analogs were similar to that of human insulin, however, aspart and lispro, but not glulisine, induced a more rapid extracellular signal-regulated kinase ERK activation through INSR-A, whereas all three analogs stimulated a more prolonged AKT activation compared to insulin [ ].
On the other hand, the long-acting analogs glargine and detemir appeared to have a low affinity for the INSR-A isoform with a longer dissociation rate and a higher mitogenic to metabolic ratio compared to native insulin [ , ]. Insulin glargine reportedly promotes the differentiation of both subcutaneous and visceral preadipocytes, as well as lipogenesis [ ]. However, these in vitro findings are somewhat difficult to reconcile with clinical data showing that some diabetic patients treated with lispro and glargine may develop lipoatrophy with severe AT inflammation [ , , ].
On the other hand, insulin detemir was shown to induce the Pparg2 adipocyte master gene to a lesser extent compared to human insulin, resulting in attenuated effects on adipocyte differentiation and lipogenesis in human subcutaneous and visceral ASCs, in spite of a similar activation of proximal insulin signaling [ 19 ]. In addition to antibodies, small synthetic peptides have been discovered that behave as INSR ligands.
For instance, an insulin mimetic peptide S displayed hypoglycemic effects in vivo but a limited mitogenic response, as well as higher lipogenesis and glucose uptake in vitro compared to native insulin [ ].
This single-chain peptide, despite binding equipotency, appeared to activate only the metabolic arm of insulin signaling i. Another ligand, S, discovered by phage display, activates INSR with sub-nanomolar affinity and exhibits agonist activity on both glucose uptake and lipogenesis [ ].
The effects of these compounds on INSR isoforms and downstream signaling remain elusive. Two INSR isoform-selective agonists were also designed by mutagenesis approaches exhibiting tissue-specific responses in a rat model: INS-A, with a strong effect on glycogen synthesis in muscle and low lipogenic activity in adipocytes and INS-B, capable of inducing a high level of lipogenesis in adipocytes and glycogen accumulation in hepatocytes [ ].
The role of insulin in regulating AT development and function is fundamental. Insulin stimulates glucose and fatty acid transport and lipid synthesis and suppresses lipolysis [ 1 ]. In addition, several molecules with paracrine and endocrine activities, such as leptin, adiponectin, chemerin, omentin and vaspin, as well as proteins involved in ECM remodeling, have an insulin-dependent regulation, and there is a complex interplay between the vascular network and the adipocytes. New synthetic and natural agonists of the INSR have been recently developed in order to improve metabolic outcomes with minor effects on mitogenesis.
Therefore, further studies will be important to design and characterize specific ligands that selectively activate more metabolically favorable responses in AT to counteract its dysfunction in obesity and T2D. National Center for Biotechnology Information , U. Int J Mol Sci. Published online Feb Author information Article notes Copyright and License information Disclaimer.
Received Jan 2; Accepted Feb 6. This article has been cited by other articles in PMC. Abstract Insulin is a major endocrine hormone also involved in the regulation of energy and lipid metabolism via the activation of an intracellular signaling cascade involving the insulin receptor INSR , insulin receptor substrate IRS proteins, phosphoinositol 3-kinase PI3K and protein kinase B AKT.
Keywords: insulin receptor, adipose tissue, adipocyte, receptor isoform. Introduction Adipose tissue AT is a critical regulator of energy balance and substrate metabolism, also through the production and secretion of several substances with endocrine or paracrine functions that are involved in energy homeostasis.
Insulin Action and AT Metabolism Insulin exerts a critical control on anabolic responses in AT by stimulating glucose and free fatty acid uptake, inhibiting lipolysis and stimulating de novo fatty acid synthesis in adipocytes Figure 1. Open in a separate window. Figure 1. Insulin Effects on AT Endocrine Activity In addition to regulating the release and storage of lipids, AT functions as a large endocrine organ that regulates several aspects of whole-body physiology through the release of hormones, lipids and cytokines adipokines [ 46 ].
Table 1 Insulin effects on adipose tissue AT endocrine activity. INSR in AT Different studies in animal models of IR conducted using the tissue-specific gene-deletion of INSR have allowed for the elucidation of the physiological roles of insulin, as well as the mechanisms underlying the development of IR in specific tissues Table 2. Conclusions The role of insulin in regulating AT development and function is fundamental.
Author Contributions V. Conflicts of Interest The authors declare no conflict of interest. References 1. Laviola L. Insulin signalling in human adipose tissue. Klip A. Cell Physiol. Rea S. Graham T. Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects. Christen T. Increased glucose uptake in visceral versus subcutaneous adipose tissue revealed by PET imaging.
JACC Cardiovasc. Perrini S. Fat depot-related differences in gene expression, adiponectin secretion, and insulin action and signalling in human adipocytes differentiated in vitro from precursor stromal cells. Wong R. Insulin signaling in fatty acid and fat synthesis: A transcriptional perspective. Merkel M. Lipoprotein lipase: Genetics, lipid uptake, and regulation. Lipid Res. Endemann G.
CD36 is a receptor for oxidized low density lipoprotein. FATP1 is an insulin-sensitive fatty acid transporter involved in diet-induced obesity. Stahl A. Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes. Petersen M.
Mechanisms of Insulin Action and Insulin Resistance. Lodhi I. Lipoexpediency: De novo lipogenesis as a metabolic signal transmitter.
Trends Endocrinol. Moreno-Navarrete J. Roberts R. Markers of de novo lipogenesis in adipose tissue: Associations with small adipocytes and insulin sensitivity in humans. Schleinitz D. The genetics of fat distribution. Song Z. Regulation and metabolic significance of de novo lipogenesis in adipose tissues. Cignarelli A. Glycolaldehyde GA is the metabolic precursor of several AGEs, and its effects vary based on food and cooking methods. Here, 3T3-L1 adipocytes were used to examine the effects of GA on obesity and insulin resistance.
We found that GA treatment did not increase lipid accumulation but increased the distribution of adipocyte differentiation. GA increased the expression of CDK2, phosphorylation of mitogen-activated protein kinases, and secretion of pro-inflammatory cytokines. Overall, these results suggest that GA can stimulate lipid metabolism, hence, we suggest that the stimulation of adipogenesis and insulin resistance by GA may be associated with the interaction between RAGE and adipogenic factors in adipocytes.
Maillard reaction is a non-enzymatic reaction between reducing sugars and amino structures in amino acids, proteins, phospholipids, or DNA and is associated with age and severity of diabetes. Following the reversible formation of Schiff bases between carbonyl and free amino groups, irreversible advanced glycation end products AGEs were formed [ 1 , 2 ].
Aldehydes, such as glycolaldehyde GA and glyceraldehyde, have also been shown to induce AGE formation. Although several studies have investigated the properties of these protein modifications, many components, including GA, have not been established in murine 3T3-L1 adipocytes. GA concentrations in adipose tissue from healthy people or patients have not been quantified so far.
Although the GA level of adipose tissue has not been quantified, its physiological concentration is estimated to range from 0. Many previous studies have been conducted in various cell models at concentrations ranging from 0. We have also checked the reasonable reasons for the various concentrations used in many previous studies.
Based on this, we performed the screening in the adipocyte cell model using concentrations ranging from 0. The production and accumulation of AGEs require large amounts of GA with steady accumulation in the adipose tissue. Therefore, we applied an acute model to study adipogenesis caused by GA-derived AGEs at the cellular level. Protein modifications through derivatization by GA may induce chronic diseases through specific receptors interaction, such as receptor for advanced glycation end products RAGE.
However, it remains unclear in GA. ROS production regulates adipocyte differentiation and is a major causing factor in diabetes through chronic lipid accumulation. During adipogenesis, in which lipid accumulation occurs, transcription factors play critical roles. In addition, adipocyte differentiation is known as an inflammatory respose, and inflammatory cytokines have been shown to be secreted through adipogenesis and mitogen-activated protein kinase MAPK phosphorylation [ 12 , 13 ].
Adipocyte differentiation processes, including the effects of ROS production, cause insulin resistance. Furthermore, mitochondrial dysfunction has been associated with type 2 Diabetes Mellitus T2DM -related insulin resistance. Glucose and lipid metabolism disorders lead to defects in insulin signaling associated with various pathological conditions [ 14 , 15 ]. Thus, the elucidation of the molecular and cellular mechanisms of underlaying insulin resistance will expand our understanding of the etiology of various diseases [ 16 ].
GA was obtained from Sigma-Aldrich St. GA was dissolved in distilled water and diluted at the indicated concentrations. This medium was replaced every 2 days. GA was maintained in the culture medium at a concentration of 0.
Following adipocyte differentiation, adipocytes were rinsed with phosphate-buffered saline PBS and fixed with 3. The fixed cells were rinsed several times with PBS and incubated with Oil Red O staining solution for 2 h at room temperature.
The dye retained in the cells was eluted with isopropanol and determined using spectrophotometric analysis at nm. The cultured cells were rinsed in PBS and suspended in a homogenizer lysis buffer. These blots were developed using an enhanced chemiluminescence kit. The cell cycle arrest was investigated using Fluorescence-activated cell sorting FACs.
Fluorescence was measured at each cycle. It is a bioluminescence assay for quantitative determination of ATP using recombinant firefly luciferase and its substrate D-luciferin.
Cells after respective treatments were rinsed with phosphate-buffered saline PBS and lysed in ATP-releasing buffer containing mM potassium phosphate buffer at pH 7. ATP concentrations in the samples were calculated from standard ATP curve and normalised to the protein content. Briefly, cells were seeded in 96 well plate and differentiated to mature adipocytes and treated as above. In principle 2-DG can be taken up by glucose transporters and metabolized to 2-DGphosphate 2-DG6P and cannot be further metabolized, and thus accumulates in the cells.
Mitochondrial membrane potential was examined by staining adipocytes with the tetraethylbenzimidazolylcarbocyanine iodide JC-1 , a cationic dye that accumulates in the normal mitochondria.
Mitochondrial membrane potential was measured using mitochondrial staining kit, JC The experiment was done as per the protocol provided with the kit JC-1 kit, Sigma. The kit uses the cationic, lipophilic dye, JC In normal cells, due to the electrochemical potential gradient, the dye concentrates in the mitochondrial matrix, where it forms red fluorescent aggregates JC-1 aggregates.
Any event that dissipates the mitochondrial membrane potential prevents the accumulation of the JC-1 dye in the mitochondria and thus, the dye is dispersed throughout the entire cell leading to a shift from red JC-1 aggregates to green fluorescence JC-1 monomers. The stain was washed off with PBS and examined under spinning disk microscope and images were collected and fluorescence intensity was also measured.
For JC-1 monomers, the fluorescence was measured at nm excitation and nm emission wavelengths, and for JC-1 aggregates, the fluorescence was measured at nm excitation and nm emission wavelengths. In addition, ROS is a critical factor that influences adipocyte differentiation and insulin resistance. In addition, when GA was treated simultaneously, the expression level of RAGE was up-regulated in a concentration-dependent manner. As shown in Fig. Adipocyte differentiation was also induced by GA treatment.
However, the amount of lipid accumulation remained unchange, while the number of differentiated cells was increased Fig. Cell lysates were collected and subjected to western blotting using antibodies against the target gene RAGE.
B ROS level was determined as described in materials and methods. Adipocytes were then stained with Oil Red O on day 7. MDI treatment. During adipocyte differentiation, adipogenic transcription factors and genes associated with adipogenesis are essential.
Therefore, we examined the effect of GA on the expressions of critical transcription factors. Furthermore, we investigated the expression levels of adipogenic factors involved in adipogenesis and accumulation through involvement in synthesis and transport of fatty acid and triglyceride.
These data indicate that GA could stimulate adipocyte differentiation through the regulation of adipogenic transcription factors and genes. Effect of glycolaldehyde on adipogenic factors during adipocyte differentiation in 3T3-L1 cells.
Protein levels were quantified using densitometric scanning. GAPDH was used as an internal control. In adipogenesis process, the mitotic clonal expansion MCE phase acts as an important factor in adipogenesis. In general, preadipocytes cultured to confluence became growth-arrested at the G0-to-G1 cell cycle during adipogenesis, and then preadipocytes re-entered the cell cycle upon induction of hormones. Therefore, we identified cell cycle factors that regulate the cell cycle at MCE phase.
MCE is an important procedure during the early phase of adipogenic differentiation. Effect of glycolaldehyde on mitotic clonal expansion during the early phase of differentiation in 3T3-L1 cells. A 3T3-L1 preadipocytes were treated in the differentiation medium containing GA 0. The percentage of cell population at each stage was determined using Kaluza Analysis Software. Protein expressions of p21, p27 and CDK2 were measured by western blot. MCE also activates several genes that cause lipid accumulation through adipogenesis.
These data indicated that the regulation of cell cycle and adipogenesis-related genes by GA are important in the early phase of adipogenesis. MAPKs are a well-known factor involved in the early stages of adipogenesis. In addition, inflammatory cytokines are excessively generated during adipogenesis in which sufficient fat is accumulated, which adversely affects adipogenesis and metabolic diseases.
MAPKs are an important factor in the regulation of metabolic disorders, such as inflammatory diseases and diabetes. The expressions and phosphorylation levels of signaling in the mechanisms under GA treatment were investigated. B Adipocytes were sustained in the differentiation medium containing the various concentrations of GA for 7 days.
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