Effects of 6-month vitamin D supplementation on insulin sensitivity and secretion: a randomised, placebo-controlled trial.

OBJECTIVE: To determine whether vitamin D3 supplementation improves insulin sensitivity, using the hyperinsulinemic-euglycemic clamp. DESIGN: This single-centre, double-blind, placebo-controlled trial randomised 96 participants at high risk of diabetes or with newly diagnosed type 2 diabetes to vitamin D3 5000 IU daily or placebo for 6 months. METHODS: We assessed at baseline and 6 months: (1) primary aim: peripheral insulin sensitivity (M-value using a 2-h hyperinsulinemic-euglycemic clamp); (2) secondary aims: other insulin sensitivity (HOMA2%S, Matsuda) and insulin secretion (insulinogenic index, C-peptide area under the curve, HOMA2-B) indices using a 2-h oral glucose tolerance test (OGTT); ß-cell function (disposition index: M-value × insulinogenic index); fasting and 2-h glucose post OGTT; HbA1c; anthropometry. RESULTS: Baseline characteristics were similar between groups (% or mean ± s.d.): women 38.5%; age 58.7 ± 9.4 years; BMI 32.2 ± 4.1 kg/m2; prediabetes 35.8%; diabetes 20.0%; 25-hydroxyvitamin D (25(OH)D) 51.1 ± 14.2 nmol/L. At 6 months, mean 25(OH)D reached 127.6 ± 26.3 nmol/L and 51.8 ± 16.5 nmol/L in the treatment and placebo groups, respectively (P < 0.001). A beneficial effect of vitamin D3 compared with placebo was observed on M-value (mean change (95% CI): 0.92 (0.24-1.59) vs -0.03 (-0.73 to 0.67); P = 0.009) and disposition index (mean change (95% CI): 267.0 (-343.4 to 877.4) vs -55.5 (-696.3 to 585.3); P = 0.039) after 6 months. No effect was seen on other outcomes. CONCLUSIONS: In individuals at high risk of diabetes or with newly diagnosed type 2 diabetes, vitamin D supplementation for 6 months significantly increased peripheral insulin sensitivity and ß-cell function, suggesting that it may slow metabolic deterioration in this population.

Reversible modulation of SIRT1 activity in a mouse strain.

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4-5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2-4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.

Hepatocyte-specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: potential role of PPARγ.

UNLABELLED: Hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)) are protected from hepatic insulin resistance evoked by high-fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6(H-KO) mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD-fed Ptpn6(f/f) counterparts. The livers from HFD-fed Ptpn6(H-KO) mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD-fed Ptpn6(H-KO) liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD-fed Ptpn6(f/f) littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up-regulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell-autonomous manner. CONCLUSION: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity.

The SIRT1 deacetylase protects mice against the symptoms of metabolic syndrome.

Type 2 diabetes, hepatic steatosis, and gut dysbiosis are pathophysiological consequences of obesity. Sirtuin (SIRT)-1 is a protein deacetylase implicated in the regulation of metabolic activity. We set out to determine whether the catalytic activity of SIRT1 plays a role in the development of metabolic syndrome, hepatic steatosis, and the distribution of gut microbiota. We challenged with a high-fat diet (HFD) a strain of mice homozygous for a Sirt1 allele carrying a point mutation that ablates the deacetylase activity of SIRT1. When compared to wild-type animals, mice lacking SIRT1 catalytic activity rapidly accumulated excessive hepatic lipid while fed the HFD, an effect evident within 2 wk of HFD feeding. Both white and brown adipose depots became hypertrophic, and the animals developed insulin resistance. The ratio of the major phyla of gut microbiota (Firmicutes and Bacteroidetes) increased rapidly in the SIRT1-deficient mice after HFD challenge. We conclude that the deacetylase activity of SIRT1 plays an important role in regulating glucose and hepatic lipid homeostasis. In addition, the composition of gut microbiota is influenced by both the animals' Sirt1 genotype and diet composition.

SIRT1 is a Highly Networked Protein That Mediates the Adaptation to Chronic Physiological Stress.

SIRT1 is a NAD(+)-dependent protein deacetylase that has a very large number of established protein substrates and an equally impressive list of biological functions thought to be regulated by its activity. Perhaps as notable is the remarkable number of points of conflict concerning the role of SIRT1 in biological processes. For example, evidence exists suggesting that SIRT1 is a tumor suppressor, is an oncogene, or has no effect on oncogenesis. Similarly, SIRT1 is variably reported to induce, inhibit, or have no effect on autophagy. We believe that the resolution of many conflicting results is possible by considering recent reports indicating that SIRT1 is an important hub interacting with a complex network of proteins that collectively regulate a wide variety of biological processes including cancer and autophagy. A number of the interacting proteins are themselves hubs that, like SIRT1, utilize intrinsically disordered regions for their promiscuous interactions. Many studies investigating SIRT1 function have been carried out on cell lines carrying undetermined numbers of alterations to the proteins comprising the SIRT1 network or on inbred mouse strains carrying fixed mutations affecting some of these proteins. Thus, the effects of modulating SIRT1 amount and/or activity are importantly determined by the genetic background of the cell (or the inbred strain of mice), and the effects attributed to SIRT1 are synthetic with the background of mutations and epigenetic differences between cells and organisms. Work on mice carrying alterations to the Sirt1 gene suggests that the network in which SIRT1 functions plays an important role in mediating physiological adaptation to various sources of chronic stress such as calorie restriction and calorie overload. Whether the catalytic activity of SIRT1 and the nuclear concentration of the co-factor, NAD(+), are responsible for modulating this activity remains to be determined. However, the effect of modulating SIRT1 activity must be interpreted in the context of the cell or tissue under investigation. Indeed, for SIRT1, we argue that context is everything.

SirT1 catalytic activity is required for male fertility and metabolic homeostasis in mice.

The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.

The proteasome inhibitor MG132 reduces immobilization-induced skeletal muscle atrophy in mice.

BACKGROUND: Skeletal muscle atrophy is a serious concern for the rehabilitation of patients afflicted by prolonged limb restriction. This debilitating condition is associated with a marked activation of NFκB activity. The ubiquitin-proteasome pathway degrades the NFκB inhibitor IκBα, enabling NFκB to translocate to the nucleus and bind to the target genes that promote muscle atrophy. Although several studies showed that proteasome inhibitors are efficient to reduce atrophy, no studies have demonstrated the ability of these inhibitors to preserve muscle function under catabolic condition. METHODS: We recently developed a new hindlimb immobilization procedure that induces significant skeletal muscle atrophy and used it to show that an inflammatory process characterized by the up-regulation of TNFα, a known activator of the canonical NFκB pathway, is associated with the atrophy. Here, we used this model to investigate the effect of in vivo proteasome inhibition on the muscle integrity by histological approach. TNFα, IL-1, IL-6, MuRF-1 and Atrogin/MAFbx mRNA level were determined by qPCR. Also, a functional measurement of locomotors activity was performed to determine if the treatment can shorten the rehabilitation period following immobilization. RESULTS: In the present study, we showed that the proteasome inhibitor MG132 significantly inhibited IκBα degradation thus preventing NFκB activation in vitro. MG132 preserved muscle and myofiber cross-sectional area by downregulating the muscle-specific ubiquitin ligases atrogin-1/MAFbx and MuRF-1 mRNA in vivo. This effect resulted in a diminished rehabilitation period. CONCLUSION: These finding demonstrate that proteasome inhibitors show potential for the development of pharmacological therapies to prevent muscle atrophy and thus favor muscle rehabilitation.

The transcription factors NFAT and CREB have different susceptibilities to the reduced Ca2+ responses caused by the knock down of inositol trisphosphate receptor in HEK 293A cells.

BACKGROUND/AIMS: The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). Three isoforms of IP(3)R have been identified and most cell types express different proportions of these isoforms. The purpose of this study was to investigate how IP(3)R signalling is involved in the activation of the Ca(2+)-sensitive transcription factors NFAT and CREB. METHODS: Each IP(3)R isoform expressed in HEK 293A cells was knocked down using selective siRNA. Free intracellular Ca(2+) was monitored spectrofluometrically. NFAT and CREB activities were measured with luciferase reporter constructs. RESULTS: IP(3)R-2-knocked down HEK 293A cells showed a deficient CCh-induced Ca(2+) response that could be rescued by co-stimulation with VIP, a cAMP increasing agonist. NFAT transcriptional activity, but not CREB transcriptional activity, was significantly reduced in IP(3)R-2-knocked down HEK 293A cells. Overexpression of IP(3)R-1 could fully compensate for IP(3)R-2 knock down to mobilize Ca(2+) and to activate NFAT. CONCLUSION: Our results show that the knock down of IP(3)R-2 significantly reduced the intracellular Ca(2+) response of HEK 293 cells. This reduced Ca(2+) response did not affect the activation of CREB but significantly decreased the activation of NFAT, suggesting that the Ca(2+) signals required for the activation of NFAT are stronger than those required for the activation of CREB.

Transcriptional profiling of skeletal muscle reveals factors that are necessary to maintain satellite cell integrity during ageing.

Skeletal muscle ageing is characterized by faulty degenerative/regenerative processes that promote the decline of its mass, strength, and endurance. In this study, we used a transcriptional profiling method to better understand the molecular pathways and factors that contribute to these processes. To more appropriately contrast the differences in regenerative capacity of old muscle, we compared it with young muscle, where robust growth and efficient myogenic differentiation is ongoing. Notably, in old mice, we found a severe deficit in satellite cells activation. We performed expression analyses on RNA from the gastrocnemius muscle of young (3-week-old) and old (24-month-old) mice. The differential expression highlighted genes that are involved in the efficient functioning of satellite cells. Indeed, the greatest number of up-regulated genes in young mice encoded components of the extracellular matrix required for the maintenance of the satellite cell niche. Moreover, other genes included Wnt inhibitors (Wif1 and Sfrp2) and Notch activator (Dner), which are putatively involved in the interconnected signalling networks that control satellite cell function. The widespread expression differences for inhibitors of TGFbeta signalling further emphasize the shortcomings in satellite cell performance. Therefore, we draw attention to the breakdown of features required to maintain satellite cell integrity during the ageing process.

A novel hindlimb immobilization procedure for studying skeletal muscle atrophy and recovery in mouse.

Skeletal muscle atrophy is a serious concern for patients afflicted by limb restriction due to surgery (e.g., arthrodesis), several articular pathologies (e.g., arthralgia), or simply following cast immobilization. To study the molecular events involved in this immobilization-induced debilitating condition, a convenient mouse model for atrophy is lacking. Here we provide a new immobilization procedure exploiting the normal flexion of the mouse hindlimb using a surgical staple to fix the ventral part of the foot to the distal part of the calf. Histological analysis revealed that our approach induced significant skeletal muscle atrophy by reducing the myofiber size of the tibialis anterior (TA) muscle by 36% compared with the untreated contralateral TA within a few days postimmobilization. Two molecular markers for atrophy, atrogin-1/muscle atrophy F-box (atrogin-1/MAFbx) and muscle ring finger 1 (MuRF-1) mRNAs, were significantly upregulated by 1.9- and 5.9-fold, respectively. Interestingly, our model also revealed the presence of an early inflammatory process during atrophy, characterized by the mRNA upregulation of TNF-alpha, IL-1, and IL-6 (1.9-, 2.4-, and 3.4-fold, respectively) simultaneously with the upregulation of the common leukocyte marker CD45 (6.1-fold). Moreover, muscle rapidly recovered on remobilization, an event associated with significantly increased levels of uncoupling protein-3 and peroxisome proliferator-activated receptor gamma coactivator-1alpha mRNA, key components of prooxidative muscle metabolism. This model offers unexpected new insights into the molecular events involved in immobilization atrophy.

Advances in myogenic cell transplantation and skeletal muscle tissue engineering.

Curative treatments are currently not available for people suffering from one of the many prevalent muscle myopathies. One approach to ameliorate these conditions relies on the cell-based transplantation of potential myogenic precursors, or more optimistically, the transfer of engineered skeletal muscle tissue. To date, clinical trials with myogenic stem cell transplantation have met with only modest success while the transplantation of engineered muscle tissue is at the earliest stages of development. Despite the slow progress, these studies have provided insights and avenues that will eventually lead to a powerful therapeutic tool.

Protein kinase C phosphorylates the inositol 1,4,5-trisphosphate receptor type 2 and decreases the mobilization of Ca2+in pancreatoma AR4-2J cells.

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor channel, which plays a major (IP(3)R) is an intracellular Ca(2+) role in Ca(2+) signalling. Three isoforms of IP(3)R have been identified (IP(3)R-1, IP(3)R-2 and IP(3)R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP(3)R are poorly understood. AR4-2J cells, which express almost exclusively (~86%) the IP(3)R-2, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) influences IP(3)R-2-mediated Ca(2+) release. Using an immunoprecipitation approach, we confirmed that AR4-2J cells express almost exclusively the IP(3)R-2 isoform. Using an in vitro phosphorylation assay, we showed that the immunopurified IP(3)R-2 was efficiently phosphorylated by exogenous PKC. In intact AR4-2J cells metabolically labelled with (32)Pi, we showed that phorbol-12-myristate-13-acetate (PMA) and Ca(2+) mobilizing agonists cause the phosphorylation of IP(3)R-2. In saponin-permeabilized AR4-2J cells, IP(3)-induced Ca(2+) release was reduced after a pre-treatment with PMA or with exogenous PKC. PMA also reduced the Ca(2+) response of intact AR4-2J cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. These results demonstrate that PKC decreases the Ca(2+)mobilizing activity of IP(3)R-2 and thus exerts a negative feedback on the agonists-induced Ca(2+) response of AR4-2J cells.

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells.

BACKGROUND INFORMATION: In endocrine cells, IP(3)R (inositol 1,4,5-trisphosphate receptor), a ligand-gated Ca2+ channel, plays an important role in the control of intracellular Ca2+ concentration. There are three subtypes of IP(3)R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP(3)R-3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP(3)R-3 in RINm5F cells. RESULTS: We show that immunoprecipitated IP(3)R-3 is a good substrate for PKA. Using a back-phosphorylation approach, we show that endogenous PKA phosphorylates IP(3)R-3 in intact RINm5F cells. [(3)H]IP(3) (inositol 1,4,5-trisphosphate) binding affinity and IP(3)-induced Ca2+ release activity were enhanced in permeabilized cells that were pre-treated with forskolin or PKA. The PKA-induced enhancement of IP(3)R-3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. CONCLUSION: The results of the present study reveal a converging step where the cAMP and the Ca2+ signalling systems act co-operatively in endocrine cell responses to external stimuli.

Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells.

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.

Synthesis of (+/-)-2-O-[4'-(N-9''-purinyl)butyl] myo-inositol 1,4,5-tris(phosphate), a potent full agonist at the D-myo-inositol 1,4,5-tris(phosphate) receptor.

Racemic 2-O-[4'(9''-N-purinyl)butyl] myo-inositol 1,4,5-tris(phosphate) 8 was synthesized starting from myo-inositol. Substitution of position 2 by an alkyl side chain was rendered possible by inversion of the chair conformation of the inositol ring by means of an orthoester. The final compound is a full agonist with the same order of potency as d-myo-inositol 1,4,5-tris(phosphate).

Expression of a truncated form of inositol 1,4,5-trisphosphate receptor type III in the cytosol of DT40 triple inositol 1,4,5-trisphosphate receptor-knockout cells.

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel playing a major role in Ca2+ signaling. Three isoforms of IP3R have been identified and most cell types express different proportions of each isoform. The DT40 B lymphocyte cell line lacking all three IP3R isoforms (DT40IP3R-KO cells) represents an excellent model to re-express any recombinant IP3R and analyze its specific properties. In the study presented here, we confirmed that DT40IP3R-KO cells do not express any IP3-sensitive Ca2+ release channel. However, with an immunoblot approach and a [3H]IP3 binding approach we demonstrated the presence of a C-terminally truncated form of IP3R type III in the cytosolic fraction of DT40IP3R-KO cells. We further showed that this truncated IP3R retained the ability to couple to the Ca2+ entry channel TRPC6. Therefore, a word of caution is offered about the interpretation of results obtained in using DT40IP3R-KO cells to study the cellular mechanisms of Ca2+ entry.

Synthesis and binding properties of cyclopentane analogues of myo-inositol 1,4,5-tris(phosphate).

Cyclopentanic analogues of myo-inositol 1,4,5-tris(phosphate) were synthesised starting from cyclopentadiene. The affinities of the trisphosphorylated derivatives for the Ins(1,4,5)P(3) receptors were equipotent to that of compound 4, showing that the relative orientation of the functional groups, particularly of the hydroxyl, is not of prime importance in this series. The (31)P NMR titration curves show that the tris(phosphate) 5 behaves as the superimposition of an independent phosphate and a vicinal bis(phosphate).

Angiotensin IV interacts with a juxtamembrane site on AT(4)/IRAP suggesting an allosteric mechanism of enzyme modulation.

Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II, binds to a specific receptor (AT(4)) that has recently been identified as the transmembrane aminopeptidase insulin-regulated aminopeptidase (IRAP) based on the fact that the two proteins share several pharmacological and biochemical properties. Our binding studies indicated that bovine heart expresses relatively large amounts (1.2 pmol/mg protein) of high-affinity binding sites for Ang IV (K(d)=1.8 nM). A photoaffinity-labeling approach combined with mild trypsin digestion revealed that the AT(4) receptor of bovine heart is a single transmembrane domain protein (153 kDa) with a large extracellular fragment (143 kDa). After alkaline denaturation of the AT(4) receptor, trypsin digestion produced two small membrane-associated fragments (16.9 and 6.6 kDa). These results suggest that Ang IV interacts with a juxtamembrane domain of AT(4) receptor. The location of the juxtamembrane site of contact was different from that of the active site of IRAP, suggesting that Ang IV uses an allosteric mechanism to modulate the activity of the AT(4)/IRAP.