Apolipoprotein A-I (apoA-I), the main protein constituent of HDL, has a central role in the reverse cholesterol-transport pathway, which together with the anti-inflammatory properties of apoA-I/HDL provide cardioprotection. Recent findings of direct stimulation of glucose uptake in muscle by apoA-I/HDL suggest that altered apoA-I and HDL functionality may be a contributing factor to the development of diabetes. We have studied the in vivo effects of short treatments with human apoA-I in a high-fat diet fed mouse model. In addition to native apoA-I, we investigated the effects of the cardioprotective Milano variant (Arg173Cys).

Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our understanding of how metabolic derangements develop in states of hyperparathyroidism, including vitamin D deficiency.

Salt-inducible kinase (SIK) 3 is a virtually unstudied, ubiquitously expressed serine/threonine kinase, belonging to the AMP-activated protein kinase (AMPK)-related family of kinases, all of which are regulated by LKB1 phosphorylation of a threonine residue in their activation (T)-loops. Findings in adrenal cells have revealed a role for cAMP in the regulation of SIK1, and recent findings suggest that insulin can regulate an SIK isoform in Drosophila. As cAMP has important functions in adipocytes, mainly in the regulation of lipolysis, we have evaluated a potential role for cAMP, as well as for insulin, in the regulation of SIK3 in these cells. We establish that raised cAMP levels in response to forskolin and the β-adrenergic receptor agonist CL 316,243 induce a phosphorylation of SIK3 in HEK293 cells and primary adipocytes. This phosphorylation coincides with increased 14-3-3 binding to SIK3 in these cell types. Our findings also show that cAMP-elevation results in reduced SIK3 activity in adipocytes. Phosphopeptide mapping and site-directed mutagenesis reveal that the cAMP-mediated regulation of SIK3 appears to depend on three residues, T469, S551 and S674, that all contribute to some extent to the cAMP-induced phosphorylation and 14-3-3-binding. As the cAMP-induced regulation can be reversed with the protein kinase A (PKA) inhibitor H89, and a role for other candidate kinases, including PKB and RSK, could be excluded, we believe that PKA is the kinase responsible for SIK3 regulation in response to elevated cAMP levels. Our findings of cAMP-mediated regulation of SIK3 suggest that SIK3 may mediate some of the effects of this important second messenger in adipocytes.

Insulin resistance is associated with oxidative stress, mitochondrial dysfunction, and a chronic low-grade inflammatory status. In this sense, cerium oxide nanoparticles (CeO2 NPs) are promising nanomaterials with antioxidant and anti-inflammatory properties. Thus, we aimed to evaluate the effect of CeO2 NPs in mouse 3T3-L1 adipocytes, RAW 264.7 macrophages, and C2C12 myotubes under control or proinflammatory conditions. Macrophages were treated with LPS, and both adipocytes and myotubes with conditioned medium (25% LPS-activated macrophages medium) to promote inflammation. CeO2 NPs showed a mean size of ≤25.3 nm (96.7%) and a zeta potential of 30.57 ± 0.58 mV, suitable for cell internalization. CeO2 NPs reduced extracellular reactive oxygen species (ROS) in adipocytes with inflammation while increased in myotubes with control medium. The CeO2 NPs increased mitochondrial content was observed in adipocytes under proinflammatory conditions. Furthermore, the expression of Adipoq and Il10 increased in adipocytes treated with CeO2 NPs. In myotubes, both Il1b and Adipoq were downregulated while Irs1 was upregulated. Overall, our results suggest that CeO2 NPs could potentially have an insulin-sensitizing effect specifically on adipose tissue and skeletal muscle. However, further research is needed to confirm these findings.

The prevalence of type 2 diabetes (T2D) is increasing worldwide, but current treatments have limitations. miRNAs may play a key role in the development of T2D and can be targets for novel therapies. Here, we examined whether T2D is associated with altered expression and DNA methylation of miRNAs using adipose tissue from 14 monozygotic twin pairs discordant for T2D. Four members each of the miR-30 and let-7-families were downregulated in adipose tissue of subjects with T2D versus control subjects, which was confirmed in an independent T2D case-control cohort. Further, DNA methylation of five CpG sites annotated to gene promoters of differentially expressed miRNAs, including miR-30a and let-7a-3, was increased in T2D versus control subjects. Luciferase experiments showed that increased DNA methylation of the miR-30a promoter reduced its transcription in vitro. Silencing of miR-30 in adipocytes resulted in reduced glucose uptake and TBC1D4 phosphorylation; downregulation of genes involved in demethylation and carbohydrate/lipid/amino acid metabolism; and upregulation of immune system genes. In conclusion, T2D is associated with differential DNA methylation and expression of miRNAs in adipose tissue. Downregulation of the miR-30 family may lead to reduced glucose uptake and altered expression of key genes associated with T2D.

Obesity and diabetes incidence rates are increasing dramatically, reaching pandemic proportions. Therefore, there is an urgent need to unravel the mechanisms underlying their pathophysiology. Of particular interest is the close interconnection between gut microbiota dysbiosis and obesity and diabetes progression. Hence, microbiota manipulation through diet has been postulated as a promising therapeutic target. In this regard, secretion of gut microbiota-derived extracellular vesicles is gaining special attention, standing out as key factors that could mediate gut microbiota-host communication. Extracellular vesicles (EVs) derived from gut microbiota and probiotic bacteria allow to encapsulate a wide range of bioactive molecules (such as/or including proteins and nucleic acids) that could travel short and long distances to modulate important biological functions with the overall impact on the host health. EV-derived from specific bacteria induce differential physiological responses. For example, a high-fat diet-induced increase of the proteobacterium Pseudomonas panacis-derived EV is closely associated with the progression of metabolic dysfunction in mice. In contrast, Akkermansia muciniphila EV are linked with the alleviation of high-fat diet-induced obesity and diabetes in mice. Here, we review the newest pieces of evidence concerning the potential role of gut microbiota and probiotic-derived EV on obesity and diabetes onset, progression, and management, through the modulation of inflammation, metabolism, and gut permeability. In addition, we discuss the role of certain dietary patterns on gut microbiota-derived EV profile and the clinical implication that dietary habits could have on metabolic diseases progression through the shaping of gut microbiota-derived EV.

AMP-activated protein kinase (AMPK) activation is considered a useful strategy for the treatment of type 2 diabetes (T2D). It is unclear whether the expression and/or activity of AMPK in adipocytes is dysregulated in obesity. Also, the expression/activity pattern of AMPKβ isoforms, which are targets for AMPK activators, in adipocytes remains elusive. In this study we show that the two AMPKβ isoforms make roughly equal contributions to AMPK activity in primary human and mouse adipocytes, whereas in cultured 3T3-L1 adipocytes of mouse origin and in primary rat adipocytes, β1-associated activity clearly dominates. Additionally, we found that obesity is not associated with changes in AMPK subunit expression or kinase activity in adipocytes isolated from subcutaneous adipose tissue from individuals with various BMI.

Obesity is linked to cardiometabolic diseases, however non-obese individuals are also at risk for type 2 diabetes (T2D) and cardiovascular disease (CVD). White adipose tissue (WAT) is known to play a role in both T2D and CVD, but the contribution of WAT inflammatory status especially in non-obese patients with cardiometabolic diseases is less understood. Therefore, we aimed to find associations between WAT inflammatory status and cardiometabolic diseases in non-obese individuals.

Salt-inducible kinase 2 (SIK2) is highly expressed in white adipocytes, but downregulated in individuals with obesity and insulin resistance. These conditions are often associated with a low-grade inflammation in adipose tissue. We and others have previously shown that SIK2 is downregulated by tumor necrosis factor α (TNFα), however, involvement of other pro-inflammatory cytokines, or the mechanisms underlying TNFα-induced SIK2 downregulation, remain to be elucidated. In this study we have shown that TNFα downregulates SIK2 protein expression not only in 3T3L1- but also in human in vitro differentiated adipocytes. Furthermore, monocyte chemoattractant protein-1 and interleukin (IL)-1β, but not IL-6, might also contribute to SIK2 downregulation during inflammation. We observed that TNFα-induced SIK2 downregulation occurred also in the presence of pharmacological inhibitors against several kinases involved in inflammation, namely c-Jun N-terminal kinase, mitogen activated protein kinase kinase 1, p38 mitogen activated protein kinase or inhibitor of nuclear factor kappa-B kinase (IKK). However, IKK may be involved in SIK2 regulation as we detected an increase of SIK2 when inhibiting IKK in the absence of TNFα. Increased knowledge about inflammation-induced downregulation of SIK2 could ultimately be used to develop strategies for the reinstalment of SIK2 expression in insulin resistance.

MicroRNAs are central players in the control of insulin secretion, but their transcriptional regulation is poorly understood. Our aim was to investigate cAMP-mediated transcriptional regulation of the miR-212/miR-132 cluster and involvement of further upstream proteins in insulin secreting β-cells. cAMP induced by forskolin+IBMX or GLP-1 caused increased expression of miR-212/miR-132, and elevated phosphorylation of cAMP-response-element-binding-protein (CREB)/Activating-transcription-factor-1 (ATF1) and Salt-Inducible-Kinases (SIKs). CyclicAMP-Regulated Transcriptional Co-activator-1 (CRTC1) was concomitantly dephosphorylated and translocated to the nucleus. Silencing of miR-212/miR-132 reduced, and overexpression of miR-212 increased, glucose-stimulated insulin secretion. Silencing of CRTC1 expression resulted in decreased insulin secretion and miR-212/miR-132 expression, while silencing or inhibition of SIKs was associated with increased expression of the microRNAs and dephosphorylation of CRTC1. CRTC1 protein levels were reduced after silencing of miR-132, suggesting feed-back regulation. Our data propose cAMP-dependent co-regulation of miR-212/miR-132, in part mediated through SIK-regulated CRTC1, as an important factor for fine-tuned regulation of insulin secretion.

In obesity, white adipose tissue (WAT) inflammation is linked to insulin resistance. Increased adipocyte chemokine (C-C motif) ligand 2 (CCL2) secretion may initiate adipose inflammation by attracting the migration of inflammatory cells into the tissue. Using an unbiased approach, we identified adipose microRNAs (miRNAs) that are dysregulated in human obesity and assessed their possible role in controlling CCL2 production. In subcutaneous WAT obtained from 56 subjects, 11 miRNAs were present in all subjects and downregulated in obesity. Of these, 10 affected adipocyte CCL2 secretion in vitro and for 2 miRNAs (miR-126 and miR-193b), regulatory circuits were defined. While miR-126 bound directly to the 3'-untranslated region of CCL2 mRNA, miR-193b regulated CCL2 production indirectly through a network of transcription factors, many of which have been identified in other inflammatory conditions. In addition, overexpression of miR-193b and miR-126 in a human monocyte/macrophage cell line attenuated CCL2 production. The levels of the two miRNAs in subcutaneous WAT were significantly associated with CCL2 secretion (miR-193b) and expression of integrin, α-X, an inflammatory macrophage marker (miR-193b and miR-126). Taken together, our data suggest that miRNAs may be important regulators of adipose inflammation through their effects on CCL2 release from human adipocytes and macrophages.

LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1-SIK pathway functions as a key gluconeogenic gatekeeper in the liver.

Dysregulation of autophagy has been observed in obesity and type 2 diabetes. Salt-inducible kinase 2 (SIK2), a member of the AMPK-related kinase family, is downregulated in adipocytes from obese or insulin resistant individuals and was previously demonstrated to regulate autophagy in cancer and normal cell lines. The aim of this study was thus to investigate a potential role of SIK2 in the regulation of adipocyte autophagy. To do so, SIK2 siRNA silencing or SIKs pharmacological inhibition of SIK2 was employed in murine differentiated 3T3-L1 adipocytes and autophagic flux was monitored. Our data indicate that SIK2 is required for both autophagic flux and expression of TFEB, the transcription factor that regulates autophagy, in adipocytes. The effect of SIK2 on autophagic flux occurs before the regulation of TFEB protein levels, suggesting different mechanisms whereby SIK2 stimulates autophagy. This study broadens the current knowledge on autophagy regulation and SIK2 function in adipocytes.

Salt-inducible kinase 2 (SIK2) is abundant in adipocytes, but downregulated in adipose tissue from individuals with obesity and insulin resistance. Moreover, SIK isoforms are required for normal insulin signalling and glucose uptake in adipocytes, but the underlying molecular mechanisms are currently not known. The adherens junction protein JUP, also termed plakoglobin or γ-catenin, has recently been reported to promote insulin signalling in muscle cells.

Caveolae are abundant adipocyte surface domains involved in insulin signaling, membrane trafficking and lipid homeostasis. Transcriptional control mechanisms for caveolins and cavins, the building blocks of caveolae, are thus arguably important for adipocyte biology and studies in this area may give insight into insulin resistance and diabetes. Here we addressed the hypothesis that one of the less characterized caveolar components, cavin-2 (SDPR), is controlled by CCAAT/Enhancer Binding Protein (CEBPα) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG). Using human mRNA expression data we found that SDPR correlated with PPARG in several tissues. This was also observed during differentiation of 3T3-L1 fibroblasts into adipocytes. Treatment of 3T3-L1-derived adipocytes with the PPARγ-activator Rosiglitazone increased SDPR and CEBPα expression at both the mRNA and protein levels. Silencing of CEBPα antagonized these effects. Further, adenoviral expression of PPARγ/CEBPα or Rosiglitazone-treatment increased SDPR expression in primary rat adipocytes. The myocardin family coactivator MKL1 was recently shown to regulate SDPR expression in human coronary artery smooth muscle cells. However, we found that actin depolymerization, known to inhibit MKL1 and MKL2, was without effect on SDPR mRNA levels in adipocytes, even though overexpression of MKL1 and MKL2 had the capacity to increase caveolins and cavins and to repress PPARγ/CEBPα. Altogether, this work demonstrates that CEBPα expression and PPARγ-activity promote SDPR transcription and further supports the emerging notion that PPARγ/CEBPα and MKL1/MKL2 are antagonistic in adipocytes.

SBI-0206965, originally identified as an inhibitor of the autophagy initiator kinase ULK1, has recently been reported as a more potent and selective AMP-activated protein kinase (AMPK) inhibitor relative to the widely used, but promiscuous inhibitor Compound C/Dorsomorphin. Here, we studied the effects of SBI-0206965 on AMPK signalling and metabolic readouts in multiple cell types, including hepatocytes, skeletal muscle cells and adipocytes. We observed SBI-0206965 dose dependently attenuated AMPK activator (991)-stimulated ACC phosphorylation and inhibition of lipogenesis in hepatocytes. SBI-0206965 (≥25 μM) modestly inhibited AMPK signalling in C2C12 myotubes, but also inhibited insulin signalling, insulin-mediated/AMPK-independent glucose uptake, and AICA-riboside uptake. We performed an extended screen of SBI-0206965 against a panel of 140 human protein kinases in vitro, which showed SBI-0206965 inhibits several kinases, including members of AMPK-related kinases (NUAK1, MARK3/4), equally or more potently than AMPK or ULK1. This screen, together with molecular modelling, revealed that most SBI-0206965-sensitive kinases contain a large gatekeeper residue with a preference for methionine at this position. We observed that mutation of the gatekeeper methionine to a smaller side chain amino acid (threonine) rendered AMPK and ULK1 resistant to SBI-0206965 inhibition. These results demonstrate that although SBI-0206965 has utility for delineating AMPK or ULK1 signalling and cellular functions, the compound potently inhibits several other kinases and critical cellular functions such as glucose and nucleoside uptake. Our study demonstrates a role for the gatekeeper residue as a determinant of the inhibitor sensitivity and inhibitor-resistant mutant forms could be exploited as potential controls to probe specific cellular effects of SBI-0206965.

Dimethylaminopurine (DMAP) has previously been used as an inhibitor of phosphorylation in studies of meiotic events, and more recently to investigate TNFalpha signaling, because of its potential to inhibit activation of c-jun N-terminal kinase (JNK). Here we have addressed the effects of DMAP on metabolic insulin responses in adipocytes and on intracellular insulin signaling molecules. At 100 micromol/L, DMAP completely inhibited the ability of insulin to counteract lipolysis in isolated adipocytes. Insulin-induced lipogenesis and glucose uptake was inhibited to a lesser degree in a concentration-dependent manner starting at 10 micromol/L DMAP. Insulin-induced tyrosine phosphorylation of the insulin receptor was not affected by DMAP. Insulin-induced activation of protein kinase B, a known mediator of insulin action, was not inhibited by 100 micromol/L, but to a low extent by 1 mmol/L DMAP in intact cells. This inhibition was not sufficient to affect activation of the downstream protein kinase B substrate phosphodiesterase 3B. The inhibition of activation of JNK as a possible mechanism whereby DMAP affects insulin-induced antilipolysis, lipogenesis, and glucose uptake, was investigated using the JNK inhibitor SP600125. At 100 micromol/L, SP600125 completely reversed the antilipolytic effect of insulin, as well as partially inhibited insulin-induced lipogenesis and glucose-uptake, indicating that JNK may be involved in mediating these actions of insulin. Inhibition of JNK by DMAP may therefore partly explain the negative impact of DMAP on insulin action in adipocytes.

White adipose tissue (WAT) morphology characterized by hypertrophy (i.e., fewer but larger adipocytes) associates with increased adipose inflammation, lipolysis, insulin resistance, and risk of diabetes. However, the causal relationships and the mechanisms controlling WAT morphology are unclear. Herein, we identified EBF1 as an adipocyte-expressed transcription factor with decreased expression/activity in WAT hypertrophy. In human adipocytes, the regulatory targets of EBF1 were enriched for genes controlling lipolysis and adipocyte morphology/differentiation, and in both humans and murine models, reduced EBF1 levels associated with increased lipolysis and adipose hypertrophy. Although EBF1 did not affect adipose inflammation, TNFα reduced EBF1 gene expression. High-fat diet intervention in Ebf1(+/-) mice resulted in more pronounced WAT hypertrophy and attenuated insulin sensitivity compared with wild-type littermate controls. We conclude that EBF1 is an important regulator of adipose morphology and fat cell lipolysis and may constitute a link between WAT inflammation, altered lipid metabolism, adipose hypertrophy, and insulin resistance.

Resistance training (RT) and n-3 polyunsaturated fatty acids (n-3 PUFA) supplementation have emerged as strategies to improve muscle function in older adults. Overweight/obese postmenopausal women (55-70 years) were randomly allocated to one of four experimental groups, receiving placebo (olive oil) or docosahexaenoic acid (DHA)-rich n-3 PUFA supplementation alone or in combination with a supervised RT-program for 16 weeks. At baseline and at end of the trial, body composition, anthropometrical measures, blood pressure and serum glucose and lipid biomarkers were analyzed. Oral glucose tolerance tests (OGTT) and strength tests were also performed. All groups exhibit a similar moderate reduction in body weight and fat mass, but the RT-groups maintained bone mineral content, increased upper limbs lean mass, decreased lower limbs fat mass, and increased muscle strength and quality compared to untrained-groups. The RT-program also improved glucose tolerance (lowering the OGTT incremental area under the curve). The DHA-rich supplementation lowered diastolic blood pressure and circulating triglycerides and increased muscle quality in lower limbs. In conclusion, 16-week RT-program improved segmented body composition, bone mineral content, and glucose tolerance, while the DHA-rich supplement had beneficial effects on cardiovascular health markers in overweight/obese postmenopausal women. No synergistic effects were observed for DHA supplementation and RT-program combination.

Human white adipose tissue (AT) is a metabolically active organ with distinct depot-specific functions. Despite their locations close to the gastrointestinal tract, mesenteric AT and epiploic AT (epiAT) have only scarcely been investigated. Here, we aim to characterise these ATs in-depth and estimate their contribution to alterations in whole-body metabolism.