Hepatocellular carcinoma induced by hepatocyte Pten deletion reduces BAT UCP-1 and thermogenic capacity in mice, despite increasing serum FGF-21 and iWAT browning

Hepatocellular carcinoma (HCC) markedly enhances liver secretion of fibroblast growth factor 21 (FGF-21), a hepatokine that increases brown and subcutaneous inguinal white adipose tissues (BAT and iWAT, respectively) uncoupling protein 1 (UCP-1) content, thermogenesis and energy expenditure. Herein, we tested the hypothesis that an enhanced BAT and iWAT UCP-1-mediated thermogenesis induced by high levels of FGF-21 is involved in HCC-associated catabolic state and fat mass reduction. For this, we evaluated body weight and composition, liver mass and morphology, serum and tissue levels of FGF-21, BAT and iWAT UCP-1 content, and thermogenic capacity in mice with Pten deletion in hepatocytes that display a well-defined progression from steatosis to steatohepatitis (NASH) and HCC upon aging. Hepatocyte Pten deficiency promoted a progressive increase in liver lipid deposition, mass, and inflammation, culminating with NASH at 24 weeks and hepatomegaly and HCC at 48 weeks of age. NASH and HCC were associated with elevated liver and serum FGF-21 content and iWAT UCP-1 expression (browning), but reduced serum insulin, leptin, and adiponectin levels and BAT UCP-1 content and expression of sympathetically regulated gene glycerol kinase (GyK), lipoprotein lipase (LPL), and fatty acid transporter protein 1 (FATP-1), which altogether resulted in an impaired whole-body thermogenic capacity in response to CL-316,243. In conclusion, FGF-21 pro-thermogenic actions in BAT are context-dependent, not occurring in NASH and HCC, and UCP-1-mediated thermogenesis is not a major energy-expending process involved in the catabolic state associated with HCC induced by Pten deletion in hepatocytes. (AU)

Chloroquine attenuates diet-induced obesity and glucose intolerance through a mechanism that might involve FGF-21, but not UCP-1-mediated thermogenesis and inhibition of adipocyte autophagy.

Chloroquine diphosphate (CQ), a weak base used to inhibit autophagic flux and treat malaria and rheumatoid diseases, has been shown, through unknown mechanisms, to improve glucose and lipid homeostasis in patients and rodents. We investigate herein the molecular mechanisms underlying these CQ beneficial metabolic actions in diet-induced obese mice. For this, C57BL6/J mice fed with either a chow or a high-fat diet (HFD) and uncoupling protein 1 (UCP-1) KO and adipocyte Atg7-deficient mice fed with a HFD were treated or not with CQ (60 mg/kg of body weight/day) during 8 weeks and evaluated for body weight, adiposity, glucose homeostasis and brown and white adipose tissues (BAT and WAT) UCP-1 content. CQ reduced body weight gain and adipose tissue and liver masses in mice fed with a HFD, without altering food intake, oxygen consumption, respiratory exchange ratio, spontaneous motor activity and feces caloric content. CQ attenuated the insulin intolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypercholesterolemia induced by HFD intake, such effects that were associated with increases in serum and liver fibroblast growth factor 21 (FGF-21) and BAT and WAT UCP-1 content. Interestingly, CQ beneficial metabolic actions of reducing body weight and adiposity and improving glucose homeostasis were preserved in HFD-fed UCP-1 KO and adipocyte Atg7 deficient mice. CQ reduces body weight gain and adiposity and improves glucose homeostasis in diet-induced obese mice through mechanisms that might involve FGF-21, but not UCP1-mediated nonshivering thermogenesis or inhibition of adipocyte autophagy.

Hepatocellular carcinoma induced by hepatocyte Pten deletion reduces BAT UCP-1 and thermogenic capacity in mice, despite increasing serum FGF-21 and iWAT browning.

Hepatocellular carcinoma (HCC) markedly enhances liver secretion of fibroblast growth factor 21 (FGF-21), a hepatokine that increases brown and subcutaneous inguinal white adipose tissues (BAT and iWAT, respectively) uncoupling protein 1 (UCP-1) content, thermogenesis and energy expenditure. Herein, we tested the hypothesis that an enhanced BAT and iWAT UCP-1-mediated thermogenesis induced by high levels of FGF-21 is involved in HCC-associated catabolic state and fat mass reduction. For this, we evaluated body weight and composition, liver mass and morphology, serum and tissue levels of FGF-21, BAT and iWAT UCP-1 content, and thermogenic capacity in mice with Pten deletion in hepatocytes that display a well-defined progression from steatosis to steatohepatitis (NASH) and HCC upon aging. Hepatocyte Pten deficiency promoted a progressive increase in liver lipid deposition, mass, and inflammation, culminating with NASH at 24 weeks and hepatomegaly and HCC at 48 weeks of age. NASH and HCC were associated with elevated liver and serum FGF-21 content and iWAT UCP-1 expression (browning), but reduced serum insulin, leptin, and adiponectin levels and BAT UCP-1 content and expression of sympathetically regulated gene glycerol kinase (GyK), lipoprotein lipase (LPL), and fatty acid transporter protein 1 (FATP-1), which altogether resulted in an impaired whole-body thermogenic capacity in response to CL-316,243. In conclusion, FGF-21 pro-thermogenic actions in BAT are context-dependent, not occurring in NASH and HCC, and UCP-1-mediated thermogenesis is not a major energy-expending process involved in the catabolic state associated with HCC induced by Pten deletion in hepatocytes.

A Family of Bisnaphthyl C2-Symmetric and Asymmetric Clefts: Synthesis, Solid-State Structure, and Calculation of the Interplanar Angle.

The synthesis of a new family of naphthalenoid C2-symmetric clefts has been realized through a four-step synthetic sequence giving three C2-symmetric clefts and a rare nonsymmetric example. Subsequently, stereoselective reduction of the carbonyl groups at C-8 and C-16 then provides cleft molecules with hydrogen bonding potential. Using single-crystal X-ray and computational analysis, the cleft angle of the dione has been determined.

Futile cycle of ß-oxidation and de novo lipogenesis are associated with essential fatty acids depletion in lipoatrophy.

Total absence of adipose tissue (lipoatrophy) is associated with the development of severe metabolic disorders including hepatomegaly and fatty liver. Here, we sought to investigate the impact of severe lipoatrophy induced by deletion of peroxisome proliferator-activated receptor gamma (PPARγ) exclusively in adipocytes on lipid metabolism in mice. Untargeted lipidomics of plasma, gastrocnemius and liver uncovered a systemic depletion of the essential linoleic (LA) and α-linolenic (ALA) fatty acids from several lipid classes (storage lipids, glycerophospholipids, free fatty acids) in lipoatrophic mice. Our data revealed that such essential fatty acid depletion was linked to increased: 1) capacity for liver mitochondrial fatty acid ß-oxidation (FAO), 2) citrate synthase activity and coenzyme Q content in the liver, 3) whole-body oxygen consumption and reduced respiratory exchange rate in the dark period, and 4) de novo lipogenesis and carbon flux in the TCA cycle. The key role of de novo lipogenesis in hepatic steatosis was evidenced by an accumulation of stearic, oleic, sapienic and mead acids in liver. Our results thus indicate that the simultaneous activation of the antagonic processes FAO and de novo lipogenesis in liver may create a futile metabolic cycle leading to a preferential depletion of LA and ALA. Noteworthy, this previously unrecognized cycle may also explain the increased energy expenditure displayed by lipoatrophic mice, adding a new piece to the metabolic regulation puzzle in lipoatrophies.

Adipocyte-specific mTORC2 deficiency impairs BAT and iWAT thermogenic capacity without affecting glucose uptake and energy expenditure in cold-acclimated mice.

Deletion of mechanistic target of rapamycin complex 2 (mTORC2) essential component rapamycin insensitive companion of mTOR (Rictor) by a Cre recombinase under control of the broad, nonadipocyte-specific aP2/FABP4 promoter impairs thermoregulation and brown adipose tissue (BAT) glucose uptake on acute cold exposure. We investigated herein whether adipocyte-specific mTORC2 deficiency affects BAT and inguinal white adipose tissue (iWAT) signaling, metabolism, and thermogenesis in cold-acclimated mice. For this, 8-wk-old male mice bearing Rictor deletion and therefore mTORC2 deficiency in adipocytes (adiponectin-Cre) and littermates controls were either kept at thermoneutrality (30 ± 1°C) or cold-acclimated (10 ± 1°C) for 14 days and evaluated for BAT and iWAT signaling, metabolism, and thermogenesis. Cold acclimation inhibited mTORC2 in BAT and iWAT, but its residual activity is still required for the cold-induced increases in BAT adipocyte number, total UCP-1 content and mRNA levels of proliferation markers Ki67 and cyclin 1 D, and de novo lipogenesis enzymes ATP-citrate lyase and acetyl-CoA carboxylase. In iWAT, mTORC2 residual activity is partially required for the cold-induced increases in multilocular adipocytes, mitochondrial mass, and uncoupling protein 1 (UCP-1) content. Conversely, BAT mTORC1 activity and BAT and iWAT glucose uptake were upregulated by cold independently of mTORC2. Noteworthy, the impairment in BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency had no major impact on whole body energy expenditure in cold-acclimated mice due to a compensatory activation of muscle shivering. In conclusion, adipocyte mTORC2 deficiency impairs, through different mechanisms, BAT and iWAT total UCP-1 content and thermogenic capacity in cold-acclimated mice, without affecting glucose uptake and whole body energy expenditure.NEW & NOTEWORTHY BAT and iWAT mTORC2 is inhibited by cold acclimation, but its residual activity is required for cold-induced increases in total UCP-1 content and thermogenic capacity, but not glucose uptake and mTORC1 activity. The impaired BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency are compensated by activation of muscle shivering in cold-acclimated mice.

PPARγ-induced upregulation of subcutaneous fat adiponectin secretion, glyceroneogenesis and BCAA oxidation requires mTORC1 activity.

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

miRNA-22 deletion limits white adipose expansion and activates brown fat to attenuate high-fat diet-induced fat mass accumulation.

BACKGROUND: Obesity, characterized by excessive expansion of white adipose tissue (WAT), is associated with numerous metabolic complications. Conversely, brown adipose tissue (BAT) and beige fat are thermogenic tissues that protect mice against obesity and related metabolic disorders. We recently reported that deletion of miR-22 enhances energy expenditure and attenuates WAT expansion in response to a high-fat diet (HFD). However, the molecular mechanisms involved in these effects mediated by miR-22 loss are unclear. METHODS AND RESULTS: Here, we show that miR-22 expression is induced during white, beige, and brown adipocyte differentiation in vitro. Deletion of miR-22 reduced white adipocyte differentiation in vitro. Loss of miR-22 prevented HFD-induced expression of adipogenic/lipogenic markers and adipocyte hypertrophy in murine WAT. In addition, deletion of miR-22 protected mice against HFD-induced mitochondrial dysfunction in WAT and BAT. Loss of miR-22 induced WAT browning. Gain- and loss-of-function studies revealed that miR-22 did not affect brown adipogenesis in vitro. Interestingly, miR-22 KO mice fed a HFD displayed increased expression of genes involved in thermogenesis and adrenergic signaling in BAT when compared to WT mice fed the same diet. CONCLUSIONS: Collectively, our findings suggest that loss of miR-22 attenuates fat accumulation in response to a HFD by reducing white adipocyte differentiation and increasing BAT activity, reinforcing miR-22 as a potential therapeutic target for obesity-related disorders.

The role of osmolytes in the temperature-triggered conformational transition of poly(N-vinylcaprolactam): an experimental and computational study.

Biomedical industries are widely exploring the use of thermo-responsive polymers (TRPs) in the advanced development of drug delivery and in many other pharmaceutical applications. There is a great need to investigate the use of less toxic and more (bio-)compatible TRPs employing several additives, which could modify the conformational transition behavior of TRPs in aqueous solution. To move forward in this aspect, we have chosen the less toxic bio-based polymer poly(N-vinylcaprolactam) (PVCL) and three different methylamine-based osmolytes, trimethylamine N-oxide (TMAO), betaine and sarcosine, in order to investigate their particular interactions with the polymer segments in PVCL and therefore the corresponding changes in the thermo-responsive conformational behavior. Several biophysical techniques, UV-visible spectroscopy, fluorescence spectroscopy, dynamic light scattering (DLS) and laser Raman spectroscopy, as well as classical computer simulation methods such as molecular dynamics are employed in the current work. All the studied methylamines are found to favor the hydrophobic collapse of the polymer thus stabilizing the globular state of PVCL. Sarcosine is observed to cause the maximum decrease in lower critical solution temperature (LCST) of PVCL followed by TMAO and then betaine. The differences observed in the LCST values of PVCL in the presence of these molecules can be attributed to the different polymer-osmolyte interactions. The less sterically hindered N atom in the case of sarcosine causes a significant difference in the phase transition temperature values of PVCL compared to betaine and TMAO, where the nitrogen atom is buried by three methyl groups attached to it.

Lipoatrophy-Associated Insulin Resistance and Hepatic Steatosis are Attenuated by Intake of Diet Rich in Omega 3 Fatty Acids.

SCOPE: Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. METHODS AND RESULTS: Severe lipoatrophic mice induced by PPAR-γ deletion exclusively in adipocytes (A-PPARγ KO) and littermate controls (A-PPARγ WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. CONCLUSION: Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Lipoatrophy-associated insulin resistance and hepatic steatosis are attenuated by intake of diet rich in omega 3 fatty acids

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-gama deletion exclusively in adipocytes (A-PPARgama KO) and littermate controls (A-PPARgama WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Lipoatrophy-associated insulin resistance and hepatic steatosis are attenuated by intake of diet rich in omega 3 fatty acids

Scope Glucose homeostasis and progression of nonalcoholic fatty liver disease (NAFLD) and hepatomegaly in severe lipoatrophic mice and their modulation by intake of a diet rich in omega 3 (n-3) fatty acids (HFO) are evaluated. Methods and results Severe lipoatrophic mice induced by PPAR-gama deletion exclusively in adipocytes (A-PPARgama KO) and littermate controls (A-PPARgama WT) are evaluated for glucose homeostasis and liver mass, proteomics, lipidomics, inflammation, and fibrosis. Lipoatrophic mice are heavier than controls, severely glucose intolerant, and hyperinsulinemic, and develop NAFLD characterized by increased liver glycogen, triacylglycerol, and diacylglycerol contents, mitotic index, apoptosis, inflammation, steatosis score, fibrosis, and fatty acid synthase (FAS) content and activity. Lipoatrophic mice also display liver enrichment with monounsaturated in detriment of polyunsaturated fatty acids including n-3 fatty acids, and increased content of cardiolipin, a tetracyl phospholipid exclusively found at the mitochondria inner membrane. Administration of a high-fat diet rich in n-3 fatty acids (HFO) to lipoatrophic mice enriches liver with n-3 fatty acids, reduces hepatic steatosis, FAS content and activity, apoptosis, inflammation, and improves glucose homeostasis. Conclusion Diet enrichment with n-3 fatty acids improves glucose homeostasis and reduces liver steatosis and inflammation without affecting hepatomegaly in severe lipoatrophic mice.

Fish Oil Protects Wild Type and Uncoupling Protein 1-Deficient Mice from Obesity and Glucose Intolerance by Increasing Energy Expenditure.

SCOPE: The mechanisms and involvement of uncoupling protein 1 (UCP1) in the protection from obesity and insulin resistance induced by intake of a high-fat diet rich in omega-3 (n-3) fatty acids are investigated. METHODS AND RESULTS: C57BL/6J mice are fed either a low-fat (control group) or one of two isocaloric high-fat diets containing either lard (HFD) or fish oil (HFN3) as fat source and evaluated for body weight, adiposity, energy expenditure, glucose homeostasis, and inguinal white and interscapular brown adipose tissue (iWAT and iBAT, respectively) gene expression, lipidome, and mitochondrial bioenergetics. HFN3 intake protected from obesity, glucose and insulin intolerances, and hyperinsulinemia. This is associated with increased energy expenditure, iWAT UCP1 expression, and incorporation of n-3 eicosapentaenoic and docosahexaenoic fatty acids in iWAT and iBAT triacylglycerol. Importantly, HFN3 is equally effective in reducing body weight gain, adiposity, and glucose intolerance and increasing energy expenditure in wild-type and UCP1-deficient mice without recruiting other thermogenic processes in iWAT and iBAT, such as mitochondrial uncoupling and SERCA-mediated calcium and creatine-driven substrate cyclings. CONCLUSION: Intake of a high-fat diet rich in omega-3 fatty acids protects both wild-type and UCP1-deficient mice from obesity and insulin resistance by increasing energy expenditure through unknown mechanisms.

PPARγ is a major regulator of branched-chain amino acid blood levels and catabolism in white and brown adipose tissues.

OBJECTIVE: We investigated whether PPARγ modulates adipose tissue BCAA metabolism, and whether this mediates the attenuation of obesity-associated insulin resistance induced by pharmacological PPARγ activation. METHODS: Mice with adipocyte deletion of one or two PPARγ copies fed a chow diet and rats fed either chow, or high fat (HF) or HF supplemented with BCAA (HF/BCAA) diets treated with rosiglitazone (30 or 15 mg/kg/day, 14 days) were evaluated for glucose and BCAA homeostasis. RESULTS: Adipocyte deletion of one PPARγ copy increased mice serum BCAA and reduced inguinal white (iWAT) and brown (BAT) adipose tissue BCAA incorporation into triacylglycerol, as well as mRNA levels of branched-chain aminotransferase (BCAT)2 and branched-chain α-ketoacid dehydrogenase (BCKDH) complex subunits. Adipocyte deletion of two PPARγ copies induced lipodystrophy, severe glucose intolerance and markedly increased serum BCAA. Rosiglitazone abolished the increase in serum BCAA induced by adipocyte PPARγ deletion. In rats, HF increased serum BCAA, such levels being further increased by BCAA supplementation. Rosiglitazone, independently of diet, lowered serum BCAA and upregulated iWAT and BAT BCAT and BCKDH activities. This was associated with a reduction in mTORC1-dependent inhibitory serine phosphorylation of IRS1 in skeletal muscle and whole-body insulin resistance evaluated by HOMA-IR. CONCLUSIONS: PPARγ, through the regulation of both BAT and iWAT BCAA catabolism in lipoeutrophic mice and muscle insulin responsiveness and proteolysis in lipodystrophic mice, is a major determinant of circulating BCAA levels. PPARγ agonism, therefore, may improve whole-body and muscle insulin sensitivity by reducing blood BCAA, alleviating mTORC1-mediated inhibitory IRS1 phosphorylation.

Molecular dynamics study of the LCST transition in aqueous poly(N-n-propylacrylamide).

The breadth of technological applications of smart polymers relies on the possibility of tuning their molecular structure to respond to external stimuli. In this context, N-substituted acrylamide-based polymers are widely studied thermoresponsive polymers. Poly(N-n-propylacrylamide) (PNnPAm), which is a structural isomer of the poly(N-isopropylacrylamide) (PNIPAm) exhibits however, a lower phase transition in aqueous solution. In this work, we use all-atom molecular dynamics simulations of PNnPAm in aqueous solutions to study, from a microscopic point-of-view, the influence of chain size and concentration on the LCST of PNnPAm. Our analysis shows that the collapse of a single oligomer of PNnPAm upon heating is dependent on the chain length and corresponds to a complex interplay between hydration and intermolecular interactions. Analysis of systems with multiple chains shows an aggregation of PNnPAm chains above the LCST.

Reply to the 'Comment on "Relating side chain organization of PNIPAm with its conformation in aqueous methanol"' by A. Pica and G. Graziano, Soft Matter, 2017, 13, DOI: 10.1039/C7SM01065F.

We have recently proposed preferential binding by a cosolvent as the mechanism for chain collapse under co-non-solvency. Here we summarise our earlier works and provide further evidence that alcohol preferentially binds to PNIPAm, forming cosolvent bridges, and thus drives the transition. We also clarify some of the common misconceptions evoked in this debate with Pica and Graziano (PG), reinforcing the arguments of our earlier reply-comment [Soft Matter, 2017, 13, 2292] and published works.

Reply to the 'Comment on "Relating side chain organization of PNIPAm with its conformation in aqueous methanol"' by N. van der Vegt and F. Rodriguez-Ropero, Soft Matter, 2017, 13, DOI: 10.1039/C6SM02139E.

In a comment van der Vegt and Rodriguez-Ropero (vdVRR) challenged our explanation of the co-non-solvency effect of PNIPAm in aqueous methanol solutions. They argue, based on a careful selection of published studies including some of their own, that direct repulsions between the different constituents are sufficient to understand this phenomenon. According to vdVRR, the emerging view of entropic collapse, put forward by Flory (1910-1985) to explain common polymers in poor solvents, would be enough to explain co-non-solvency. In this reply we attempt to bring this discussion into firmer grounds. We provide a more comprehensive view of available experimental, numerical and theoretical results and review basic concepts of physical chemistry and of statistical mechanics of polymer collapse that show how methanol mediated attractions between chain monomers are required to understand this fascinating behavior.

Effects of stereochemistry and copolymerization on the LCST of PNIPAm.

Poly(N-isopropylacrylamide) (PNIPAm) is a smart polymer that presents a lower critical transition temperature (LCST) of 305 K. Interestingly, this transition point falls within the range of the human body temperature, making PNIPAm a highly suitable candidate for bio-medical applications. However, it is sometimes desirable to have a rather flexible tuning of the LCST of these polymers to further increase their range of applications. In this work, we use all-atom molecular dynamics simulations to study the LCST of PNIPAm-based (co-)polymers. We study different molecular architectures where the polymer sequences are tuned either by modifying its stereochemistry or by the co-polymerization of PNIPAm with acrylamide (Am) units. Our analysis connects global polymer conformations with the microscopic intermolecular interactions. These findings suggest that the collapse of a PNIPAm chain upon heating is dependent on the hydration structure around the monomers, which is strongly dependent on the tacticity and the presence of more hydrophilic acrylamide monomers. Our results are found to be in good agreement with the existing experimental data.

Relating side chain organization of PNIPAm with its conformation in aqueous methanol.

Combining nuclear magnetic resonance (NMR), dynamic light scattering (DLS), and µs long all-atom simulations with two million particles, we establish a delicate correlation between increased side chain organization of PNIPAm and its collapse in aqueous methanol mixtures. We find that the preferential binding of methanol with PNIPAm side chains, bridging distal monomers along the polymer backbone, results in increased organization. Furthermore, methanol-PNIPAm preferential binding is dominated by hydrogen bonding. Our findings reveal that the collapse of PNIPAm is dominated by enthalpic interactions and that the standard poor solvent (entropic) effects play no major role.