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The adipose tissue stores excess energy in the form of neutral lipids within adipocyte lipid droplets (LDs). The correct function of LDs requires the interaction with other organelles, such as the endoplasmic reticulum (ER) as well as with LD coat-associated proteins, including Rab18, a mediator of intracellular lipid trafficking and ER–LD interaction. Although perturbations of the inter-organelle contact sites have been linked to several diseases, such as cancer, no information regarding ER–LD contact sites in dysfunctional adipocytes from the obese adipose tissue has been published to date. Herein, the ER–LD connection and Rab18 distribution at ER–LD contact sites are examined in adipocytes challenged with fibrosis and inflammatory conditions, which represent known hallmarks of the adipose tissue in obesity. Our results show that adipocytes differentiated in fibrotic conditions caused ER fragmentation, the expansion of ER–LD contact sites, and modified Rab18 dynamics. Likewise, adipocytes exposed to inflammatory conditions favored ER–LD contact, Rab18 accumulation in the ER, and Rab18 redistribution to large LDs. Finally, our studies in human adipocytes supported the suggestion that Rab18 transitions to the LD coat from the ER. Taken together, our results suggest that obesity-related pathogenic processes alter the maintenance of ER–LD interactions and interfere with Rab18 trafficking through these contact sites.

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane‐binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled‐coil protein‐2 ( NECC 2), in adipocytes. Studies in 3T3‐L1 cells showed that NECC 2 expression increased during adipogenesis. Furthermore, NECC 2 co‐immunoprecipitated with caveolin‐1 ( CAV 1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV 1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC 2 overexpression enhanced insulin‐activated Akt phosphorylation, whereas NECC 2 downregulation impaired insulin‐induced phosphorylation of Akt and ERK 2. Finally, an up‐regulation of NECC 2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3‐L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC 2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.

Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we aimed at identifying the pathogenic processes underlying impaired adipocyte differentiation in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). We report that down-regulation of a key member of the major spliceosome, PRFP8 /PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8 /PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of endoplasmic reticulum (ER)-associated protein degradation (ERAD) hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals.

The energetic demands of proliferating cells during tumorigenesis require close coordination between the cell cycle and metabolism. While CDK4 is known for its role in cell proliferation, its metabolic function in cancer, particularly in triple-negative breast cancer (TNBC), remains unclear. Our study, using genetic and pharmacological approaches, reveals that CDK4 inactivation only modestly impacts TNBC cell proliferation and tumor formation. Notably, CDK4 depletion or long-term CDK4/6 inhibition confers resistance to apoptosis in TNBC cells. Mechanistically, CDK4 enhances mitochondria-endoplasmic reticulum contact (MERCs) formation, promoting mitochondrial fission and ER-mitochondrial calcium signaling, which are crucial for TNBC metabolic flexibility. Phosphoproteomic analysis identified CDK4’s role in regulating PKA activity at MERCs. In this work, we highlight CDK4’s role in mitochondrial apoptosis inhibition and suggest that targeting MERCs-associated metabolic shifts could enhance TNBC therapy. CDK4/6 inhibitors (CDK4/6i) have improved cancer patient outcomes but shown limited benefits for those with triple-negative breast cancer (TNBC). Here, the authors report that CDK4/6 inhibition prevents CDK4 enhanced mitochondria-endoplasmic reticulum interactions, inhibiting mitochondrial apoptosis and driving resistance to CDK4/6i in TNBC models.

Background Excessive lipid accumulation in the adipose tissue in obesity alters the endocrine and energy storage functions of adipocytes. Adipocyte lipid droplets represent key organelles coordinating lipid storage and mobilization in these cells. Recently, we identified the small GTPase, Rab34, in the lipid droplet proteome of adipocytes. Herein, we have characterized the distribution, intracellular transport, and potential contribution of this GTPase to adipocyte physiology and its regulation in obesity. Methods 3T3-L1 and human primary preadipocytes were differentiated in vitro and Rab34 distribution and trafficking were analyzed using markers of cellular compartments. 3T3-L1 adipocytes were transfected with expression vectors and/or Rab34 siRNA and assessed for secretory activity, lipid accumulation and expression of proteins regulating lipid metabolism. Proteomic and protein interaction analyses were employed for the identification of the Rab34 interactome. These studies were combined with functional analysis to unveil the role played by the GTPase in adipocytes, with a focus on the actions conveyed by Rab34 interacting proteins. Finally, Rab34 regulation in response to obesity was also evaluated. Results Our results show that Rab34 localizes at the Golgi apparatus in preadipocytes. During lipid droplet biogenesis, Rab34 translocates from the Golgi to endoplasmic reticulum-related compartments and then reaches the surface of adipocyte lipid droplets. Rab34 exerts distinct functions related to its intracellular location. Thus, at the Golgi, Rab34 regulates cisternae integrity as well as adiponectin trafficking and oligomerization. At the lipid droplets, this GTPase controls lipid accumulation and lipolysis through its interaction with the E1-ubiquitin ligase, UBA1, which induces the ubiquitination and proteasomal degradation of the fatty acid transporter and member of Rab34 interactome, FABP5. Finally, Rab34 levels in the adipose tissue and adipocytes are regulated in response to obesity and related pathogenic insults (i.e., fibrosis). Conclusions Rab34 plays relevant roles during adipocyte differentiation, including from the regulation of the oligomerization (i.e., biological activity) and secretion of a major adipokine with insulin-sensitizing actions, adiponectin, to lipid storage and mobilization from lipid droplets. Rab34 dysregulation in obesity may contribute to the altered adipokine secretion and lipid metabolism that characterize adipocyte dysfunction in conditions of excess adiposity.

Adipocyte dysfunction in obesity is commonly associated with impaired insulin signalling in adipocytes and insulin resistance. Insulin signalling has been associated with caveolae, which are coated by large complexes of caveolin and cavin proteins, along with proteins with membrane‐binding and remodelling properties. Here, we analysed the regulation and function of a component of caveolae involved in growth factor signalling in neuroendocrine cells, neuroendocrine long coiled‐coil protein‐2 (NECC2), in adipocytes. Studies in 3T3‐L1 cells showed that NECC2 expression increased during adipogenesis. Furthermore, NECC2 co‐immunoprecipitated with caveolin‐1 (CAV1) and exhibited a distribution pattern similar to that of the components of adipocyte caveolae, CAV1, Cavin1, the insulin receptor and cortical actin. Interestingly, NECC2 overexpression enhanced insulin‐activated Akt phosphorylation, whereas NECC2 downregulation impaired insulin‐induced phosphorylation of Akt and ERK2. Finally, an up‐regulation of NECC2 in subcutaneous and omental adipose tissue was found in association with human obesity and insulin resistance. This effect was also observed in 3T3‐L1 adipocytes exposed to hyperglycaemia/hyperinsulinemia. Overall, the present study identifies NECC2 as a component of adipocyte caveolae that is regulated in response to obesity and associated metabolic complications, and supports the contribution of this protein as a molecular scaffold modulating insulin signal transduction at these membrane microdomains.

Background: Obesity is characterized by adipose tissue dysregulation and predisposes individuals to insulin resistance and type 2 diabetes. At the molecular level, adipocyte dysfunction has been linked to obesity-triggered oxidative stress and protein carbonylation, considering protein carbonylation as a link between oxidative stress and metabolic dysfunction. The identification of specific carbonylated proteins in adipose tissue could provide novel biomarkers of oxidative damage related to metabolic status (i.e prediabetes). Thus, we aimed at characterizing the subcutaneous and omental human adipose tissue carbonylome in obesity-associated insulin resistance. Methods: 2D-PAGE was used to identify carbonylated proteins, and clinical correlations studies and molecular biology approaches including intracellular trafficking, reactive oxygen species assay, and iron content were performed using in vitro models of insulin resistance. Results: The carbonylome of human adipose tissue included common (serotransferrin, vimentin, actin, and annexin A2) and depot-specific (carbonic anhydrase and α-crystallin B in the subcutaneous depot; and α-1-antitrypsin and tubulin in the omental depot) differences that point out the complexity of oxidative stress at the metabolic level, highlighting changes in carbonylated transferrin expression. Posterior studies using in vitro prediabetic model evidence alteration in transferrin receptor translocation, linked to the prediabetic environment. Finally, ligand-receptor molecular docking studies showed a reduced affinity for carbonylated transferrin binding to its receptor compared to wild-type transferrin, emphasizing the role of transferrin carbonylation in the link between oxidative stress and metabolic dysfunction. Conclusions: The adipose tissue carbonylome contributes to understanding the molecular mechanism driving adipocyte dysfunction and identifies possible adipose tissue carbonylated targets in obesity-associated insulin resistance.

The shift in the energetic demands of proliferating cells during tumorigenesis requires intense crosstalk between the cell cycle and metabolism. Beyond their role in cell proliferation, cell cycle regulators also modulate intracellular metabolism in normal tissues. However, in the context of cancer, where CDK4 is upregulated or stabilized, the metabolic role of CDK4 is barely understood. Using both genetic and pharmacological approaches, we aimed to determine the metabolic role of CDK4 in TNBC cells. Unexpectedly, deletion of CDK4 only slightly reduced triple-negative breast cancer (TNBC) cell proliferation and allowed tumor formation in vivo. Furthermore, proapoptotic stimuli failed to induce appropriate cell death in TNBC cells with CDK4 depletion or long-term CDK4/6 inhibitor treatment. Mechanistically, CDK4 enhances mitochondria-ER contact (MERC) formation, thus promoting mitochondrial fission and ER-mitochondrial calcium signaling. Phosphoproteomic analysis also revealed a role for CDK4 in regulating PKA activity at MERCs to sustain ER-mitochondrial calcium signaling. This proper CDK4-mediated mitochondrial calcium signaling is then required for metabolic flexibility of TNBC cells. Taken together, these results demonstrate that CDK4 inhibition leads to cell death resistance, inhibiting mitochondrial apoptosis and functions through attenuated MERCs formation and ER-mitochondrial calcium signaling in TNBC. Overall, this study provides new insights into the mechanisms of TNBC resistance to CDK4/6i therapy and paves the way to explore potential synergistic therapeutic targeting MERCs-associated metabolic shifts.

Olfactomedin-2 (OLFM2) is a pleiotropic glycoprotein emerging as a regulator of energy homeostasis. We here show the expression of OLFM2 to be adipocyte-specific and inversely associated with obesity. OLFM2 levels increase during adipogenesis and are suppressed in inflamed adipocytes. Functionally, OLFM2 deficiency impairs adipocyte differentiation, while its over-production enhances the adipogenic transformation of fat cell progenitors. Loss and gain of function experiments revealed that OLFM2 modulates key metabolic and structural pathways, including PPAR signaling, citrate cycle, fatty acid degradation, axon guidance and focal adhesion in 3T3 cell lines and primary human adipocytes. On the molecular level, OLFM2 deficiency in differentiated adipocytes predominantly downregulates genes involved in cell cycle. Extending these findings in vivo, both whole-body Olfm2 knockout and adipose-specific Olfm2 depletion in mice resulted in impaired adipose cell cycle gene expression, with the latter also displaying fat mass accretion and metabolic dysfunction. Collectively, our results underscore a critical role for OLFM2 in adipocyte biology, and support a causative link between reduced adipose OLFM2 and the pathophysiology of obesity. Olfactomedin-2 is a pleiotropic glycoprotein emerging as a regulator of energy homeostasis via the hypothalamus. The present findings functionally connect adipose-specific OLFM2 to obesity, and highlight its significance in maintaining adipocyte commitment to avoid metabolic disease.

Preadipocytes are crucial for healthy adipose tissue expansion. Preadipocyte differentiation is altered in obese individuals, which has been proposed to contribute to obesity-associated metabolic disturbances. Here, we demonstrate that impaired alternative splicing and dysregulated endoplasmic reticulum (ER)-associated protein degradation (ERAD) represent marker pathways of dysfunctional preadipocytes in obese individuals with insulin resistance (IR)/type 2 diabetes (T2D). Down-regulation of a key member of the major spliceosome, PRFP8/PRP8, as observed in IR/T2D preadipocytes from subcutaneous (SC) fat, prevented adipogenesis by altering both the expression and splicing patterns of adipogenic transcription factors and lipid droplet-related proteins, while adipocyte differentiation was restored upon recovery of PRFP8/PRP8 normal levels. Adipocyte differentiation was also compromised under conditions of ERAD hyperactivation, as occurs in SC and omental (OM) preadipocytes in IR/T2D obesity. Thus, targeting mRNA splicing and ER proteostasis in preadipocytes could improve adipose tissue function and thus contribute to metabolic health in obese individuals. Competing Interest Statement The authors have declared no competing interest.