Low 24-hour core body temperature as a thrifty metabolic trait driving catch-up fat during weight regain after caloric restriction.

The recovery of body weight after substantial weight loss or growth retardation is often characterized by a disproportionately higher rate of fat mass vs. lean mass recovery, with this phenomenon of "preferential catch-up fat" being contributed by energy conservation (thrifty) metabolism. To test the hypothesis that a low core body temperature (Tc) constitutes a thrifty metabolic trait underlying the high metabolic efficiency driving catch-up fat, the Anipill system, with telemetry capsules implanted in the peritoneal cavity, was used for continuous monitoring of Tc for several weeks in a validated rat model of semistarvation-refeeding in which catch-up fat is driven solely by suppressed thermogenesis. In animals housed at 22°C, 24-h Tc was reduced in response to semistarvation (-0.77°C, P < 0.001) and remained significantly lower than in control animals during the catch-up fat phase of refeeding (-0.27°C on average, P < 0.001), the lower Tc during refeeding being more pronounced during the light phase than during the dark phase of the 24-h cycle (-0.30°C vs. -0.23°C, P < 0.01) and with no between-group differences in locomotor activity. A lower 24-h Tc in animals showing catch-up fat was also observed when the housing temperature was raised to 29°C (i.e., at thermoneutrality). The reduced energy cost of homeothermy in response to caloric restriction persists during weight recovery and constitutes a thrifty metabolic trait that contributes to the high metabolic efficiency that underlies the rapid restoration of the body's fat stores during weight regain, with implications for obesity relapse after therapeutic slimming and the pathophysiology of catch-up growth.

Countering impaired glucose homeostasis during catch-up growth with essential polyunsaturated fatty acids: is there a major role for improved insulin sensitivity?

BACKGROUND/OBJECTIVES: Catch-up growth, an important risk factor for later obesity and type 2 diabetes, is often characterized by a high rate of fat deposition associated with hyperinsulinemia and glucose intolerance. We tested here the hypothesis that refeeding on a high-fat diet rich in essential polyunsaturated fatty acids (ePUFA) improves glucose homeostasis primarily by enhancing insulin sensitivity in skeletal muscles and adipose tissues. METHODS: Rats were caloric restricted for 2 weeks followed by 1-2 weeks of isocaloric refeeding on either a low-fat (LF) diet, a high-fat (HF) diet based on animal fat and high in saturated and monounsaturated fatty acids (HF SMFA diet), or a HF diet based on vegetable oils (1:1 mixture of safflower and linseed oils) and rich in the essential fatty acids linoleic and α-linolenic acids (HF ePUFA diet). In addition to measuring body composition and a test of glucose tolerance, insulin sensitivity was assessed during hyperinsulinemic-euglycemic clamps at the whole-body level and in individual skeletal muscles and adipose tissue depots. RESULTS: Compared to animals refed the LF diet, those refed the HF-SMFA diet showed a higher rate of fat deposition, higher plasma insulin and glucose responses during the test of glucose tolerance, and markedly lower insulin-stimulated glucose utilization at the whole body level (by a-third to a-half) and in adipose tissue depots (by 2-5 folds) during insulin clamps. While refeeding on the ePUFA diet prevented the increases in fat mass and in plasma insulin and glucose, the results of insulin clamps revealed that insulin-stimulated glucose utilization was not increased in skeletal muscles and only marginally higher in adipose tissues and at the whole-body level. CONCLUSIONS: These results suggest only a minor role for enhanced insulin sensitivity in the mechanisms by which diets high in ePUFA improves glucose homeostasis during catch-up growth.

Substantial Inter-Subject Variability in Blood Pressure Responses to Glucose in a Healthy, Non-obese Population.

Aim: A large inter-subject variability in the blood pressure (BP) response to glucose drinks has been reported. However, the underlying factors remain elusive and we hypothesized that accompanying changes in glucose metabolism affect these BP responses. Methods: Cardiovascular and glycemic changes in response to a standard 75 g oral-glucose-tolerance-test were investigated in 30 healthy, non-obese males. Continuous cardiovascular monitoring, including beat-to-beat BP, electrocardiographically deduced heart rate and impedance cardiography, was performed during a 30 min baseline and continued up to 120 min after glucose ingestion. Blood samples were taken at baseline, 30, 60, 90, and 120 min for the assessment of glucose, insulin and c-peptide. Additionally, we evaluated body composition by using validated bioelectrical impedance techniques. Results: Individual overall changes (i.e., averages over 120 min) for systolic BP ranged from -4.9 to +4.7 mmHg, where increases and decreases were equally distributed (50%). Peak changes (i.e., peak averages over 10 min intervals) for systolic BP ranged from -1.3 to +9.5 mmHg, where 93% of subjects increased systolic BP above baseline values (similar for diastolic BP) whilst 63% of subjects increased peak systolic BP by more than 4 mmHg. Changes in peak systolic BP were negatively associated with the calculated Matsuda-index of insulin sensitivity (r = -0.39, p = 0.04) but with no other evaluated parameter including body composition. Moreover, besides a trend toward an association between overall changes in systolic BP and total fat mass percentage (r = +0.32, p = 0.09), no association was found between other body composition parameters and overall BP changes. Conclusion: Substantial inter-subject variability in BP changes was observed in a healthy, non-obese subpopulation in response to an oral glucose load. In 63% of subjects, peak systolic BP increased by more than a clinically relevant 4 mmHg. Peak systolic BP changes, but not overall BP changes, correlated with insulin sensitivity, with little influence of body composition.

Gap junctional communication in tissue inflammation and repair.

Local injury induces a complex orchestrated response to stimulate healing of injured tissues, cellular regeneration and phagocytosis. Practically, inflammation is defined as a defense process whereby fluid and white blood cells accumulate at a site of injury. The balance of cytokines, chemokines, and growth factors is likely to play a key role in regulating important cell functions such as migration, proliferation, and matrix synthesis during the process of inflammation. Hence, the initiation, maintenance, and resolution of innate responses depend upon cellular communication. A process similar to tissue repair and subsequent scarring is found in a variety of fibrotic diseases. This may occur in a single organ such as liver, kidneys, pancreas, lung, skin, and heart, but fibrosis may also have a more generalized distribution such as in atherosclerosis. The purpose of this review is to summarize recent advances on the contribution of gap junction-mediated intercellular communication in the modulation of the inflammatory response and tissue repair.

Gap junctional communication does not contribute to the interaction between neutrophils and airway epithelial cells.

Cystic fibrosis (CF) is characterized by intense neutrophil migration into the airways. Increasing evidence indicates that interaction between neutrophils and airway epithelial cells contributes to the modulation of the inflammatory response. Blood neutrophils were reported to express connexins and form gap junctions with endothelial cells, thereby establishing gap junctional communication. We tested whether altered communication between human neutrophils and airway epithelial cells may contribute to the exaggerated inflammatory response observed in CF patients. Microinjections did not reveal dye coupling between activated blood neutrophils. By contrast, diffusion of calcein between neutrophils and airway epithelial cells of CF or non-CF origin was observed in transmigration and adhesion assays. This diffusion was prevented with probenicid, an inhibitor of ATP-dependent organic anion pumps, but not with gap junction blockers. Finally, RT-PCR failed to detect mRNAs for six connexins in blood neutrophils. These results suggest that gap junctional communication does not contribute to neutrophil-airway epithelial cell interaction.

Role of the carboxyl terminal of connexin43 in transjunctional fast voltage gating.

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.

Arginase II Promotes Macrophage Inflammatory Responses Through Mitochondrial Reactive Oxygen Species, Contributing to Insulin Resistance and Atherogenesis.

BACKGROUND: Macrophage-mediated chronic inflammation is mechanistically linked to insulin resistance and atherosclerosis. Although arginase I is considered antiinflammatory, the role of arginase II (Arg-II) in macrophage function remains elusive. This study characterizes the role of Arg-II in macrophage inflammatory responses and its impact on obesity-linked type II diabetes mellitus and atherosclerosis. METHODS AND RESULTS: In human monocytes, silencing Arg-II decreases the monocytes' adhesion to endothelial cells and their production of proinflammatory mediators stimulated by oxidized low-density lipoprotein or lipopolysaccharides, as evaluated by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Macrophages differentiated from bone marrow cells of Arg-II-deficient (Arg-II(-/-)) mice express lower levels of lipopolysaccharide-induced proinflammatory mediators than do macrophages of wild-type mice. Importantly, reintroducing Arg-II cDNA into Arg-II(-/-) macrophages restores the inflammatory responses, with concomitant enhancement of mitochondrial reactive oxygen species. Scavenging of reactive oxygen species by N-acetylcysteine prevents the Arg-II-mediated inflammatory responses. Moreover, high-fat diet-induced infiltration of macrophages in various organs and expression of proinflammatory cytokines in adipose tissue are blunted in Arg-II(-/-) mice. Accordingly, Arg-II(-/-) mice reveal lower fasting blood glucose and improved glucose tolerance and insulin sensitivity. Furthermore, apolipoprotein E (ApoE)-deficient mice with Arg-II deficiency (ApoE(-/-)Arg-II(-/-)) display reduced lesion size with characteristics of stable plaques, such as decreased macrophage inflammation and necrotic core. In vivo adoptive transfer experiments reveal that fewer donor ApoE(-/-)Arg-II(-/-) than ApoE(-/-)Arg-II(+/+) monocytes infiltrate into the plaque of ApoE(-/-)Arg-II(+/+) mice. Conversely, recipient ApoE(-/-)Arg-II(-/-) mice accumulate fewer donor monocytes than do recipient ApoE(-/-)Arg-II(+/+) animals. CONCLUSIONS: Arg-II promotes macrophage proinflammatory responses through mitochondrial reactive oxygen species, contributing to insulin resistance and atherogenesis. Targeting Arg-II represents a potential therapeutic strategy in type II diabetes mellitus and atherosclerosis. (J Am Heart Assoc. 2012;1:e000992 doi: 10.1161/JAHA.112.000992.).

Hyperactive S6K1 mediates oxidative stress and endothelial dysfunction in aging: inhibition by resveratrol.

Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20-24 months) as compared to the young animals (1-3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease.

Functional expression of connexin30 and connexin31 in the polarized human airway epithelium.

Gap junctions are documented in the human airway epithelium but the functional expression and molecular identity of their protein constituents (connexins, Cx) in the polarized epithelium is not known. To address this question, we documented the expression of a family of epithelial Cx (Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx37, Cx40, and Cx43) in primary human airway epithelial cells (AEC) grown on porous supports. Under submerged conditions, AEC formed a monolayer of airway cells whereas the air-liquid interface induced within 30-60 days AEC differentiation into a polarized epithelium for up to 6-9 months. Maturation of AEC was associated with the down-regulation of Cx26 and Cx43. The well-differentiated airway epithelium exhibited gap junctional communication between ciliated and between ciliated and basal cells. Interestingly, Cx30 was mostly present between ciliated cells whereas Cx31 was found between basal cells. These results are supportive of the establishment of signal-selective gap junctions with maturation of AEC, likely contributing to support airway epithelium function. These results lay the ground for studying the role of Cx-mediated cell-cell communication during repair following AEC injury and exploring Cx-targeted interventions to modulate the healing process.

A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover.

The gap junction protein connexin43 is known to have a rapid turnover, involving degradation by both the proteasomal and lysosomal systems, but the structural features of connexin43 that govern these actions are not known. The connexin43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein-protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxphi; =hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins. As both motifs may control turnover of connexin43, we used a combination of metabolic radiolabelling, immuno-precipitation and functional assays to determine the possible role of these motifs in controlling degradation of human connexin43 expressed in SKHep1 cells. Mutation V289D in the tyrosine-based sorting motif increased the steady-state pool of connexin43 by approximately 3.5-fold, while mutation P283L in the PY-motif produced a comparatively modest augmentation (1.7-fold). No additive effect was observed when the overlapping tyrosine was mutated. In pulse-chase experiments, the Y286A substitution increased the half-life of connexin43 from 2 to 6 hours, indicating that the increased steady-state levels reflected reduced protein degradation. Moreover, expression at the junctional membrane, as well as gap junction-mediated intercellular communication (GJC), were nearly abolished by lysosomal inhibitors and Brefeldin A in cells expressing wild-type connexin43, but were unaffected in the tyrosine mutant. These results provide strong evidence that the tyrosine-based motif of human connexin43 is a prime determinant controlling connexin43 stability, and consequently GJC, by targeting connexin43 for degradation in the endocytic/lysosomal compartment.

Defective activation of c-Src in cystic fibrosis airway epithelial cells results in loss of tumor necrosis factor-alpha-induced gap junction regulation.

Tumor necrosis factor-alpha (TNF-alpha) signaling is central to the transmission of the innate immune response and subsequent activation of the adaptive immune system. The functioning of both systems is required for optimal clearance of pathogens from the airways. In cystic fibrosis (CF), dysfunction of the CF transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections despite an intense inflammatory and immune response. We reported recently that TNF-alpha decreased gap junction connectivity in non-CF airway cells, a mechanism that was absent in CF cells expressing the DeltaPhe-508 mutant of CFTR. We have now identified the tyrosine kinase c-Src as a possible pathway between the mediators of inflammation and the gap junction protein connexin43 (Cx43). Indeed, TNF-alpha increased the proportion of activated c-Src in non-CF airway cells. Moreover, pharmacological antagonists and expression in non-CF cells of a dominant negative construct of c-Src prevented Cx43 channel closure by TNF-alpha. Finally, gap junction channel closure was prevented by expression of a Cx43 mutant lacking tyrosine phosphorylation sites for c-Src. Additional experiments showed that activation of c-Src was defective in CF airway cells but rescued in CFTR-corrected CF cells. These data suggest that CFTR dysfunction is associated with altered TNF-alpha signaling, resulting in the persistence of gap junction connectivity in CF airway cells. We propose that altered regulation of c-Src may contribute to the dysregulated inflammatory response that is characteristic of the CF phenotype.