In addition to signaling through the classical tyrosine kinase pathway, recent studies indicate that insulin receptors (IRs) and insulin-like growth factor 1 (IGF1) receptors (IGF1Rs) can emit signals in the unoccupied state through some yet-to-be-defined noncanonical pathways. Here we show that cells lacking both IRs and IGF1Rs exhibit a major decrease in expression of multiple imprinted genes and microRNAs, which is partially mimicked by inactivation of IR alone in mouse embryonic fibroblasts or in vivo in brown fat in mice. This down-regulation is accompanied by changes in DNA methylation of differentially methylated regions related to these loci. Different from a loss of imprinting pattern, loss of IR and IGF1R causes down-regulated expression of both maternally and paternally expressed imprinted genes and microRNAs, including neighboring reciprocally imprinted genes. Thus, the unoccupied IR and IGF1R generate previously unidentified signals that control expression of imprinted genes and miRNAs through transcriptional mechanisms that are distinct from classical imprinting control.

UNLABELLED: The aim of this study was to assess the volume and function of human brown adipose tissue (BAT) in vivo using MR imaging. METHODS: BAT volumes under thermoneutral conditions in the cervical areas were assessed via water-fat contrast using the Dixon method and via water-saturation efficiency using fast spin-echo and T2-weighted images. The existence of cervical BAT was also assessed by (18)F-FDG PET/CT scans in the same subjects. BAT functionality was assessed via functional MR imaging (fMRI) blood oxygenation level-dependent (BOLD) signal changes in response to a mild cold challenge. RESULTS: Under thermoneutral conditions, we were able to distinguish BAT from white adipose tissue in the cervical and supraclavicular fat. BAT showed higher water-to-fat contrast and higher water-saturation efficiency in MR imaging scans. The location and volume of BAT assessed by MR imaging were comparable to the measurements by (18)F-FDG PET/CT scans. During mild cold challenge, BOLD fMRI signal increased in BAT by 10.7% ± 1.8% (P 0.01). CONCLUSION: We demonstrated the feasibility of using MR imaging and fMRI to assess BAT volume and BAT responses to mild cold stimulation in the cervical areas of human subjects.

Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ~2% of spermatozoa. Thus, different "adipocyte-specific" Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.

BACKGROUND: CEL-MODY is a monogenic form of diabetes with exocrine pancreatic insufficiency caused by mutations in CARBOXYL-ESTER LIPASE (CEL). The pathogenic processes underlying CEL-MODY are poorly understood, and the global knockout mouse model of the CEL gene (CELKO) did not recapitulate the disease. We therefore aimed to create and phenotype a mouse model specifically over-expressing mutated CEL in the pancreas. METHODS: We established a monotransgenic floxed (flanking LOX sequences) mouse line carrying the human CEL mutation c.1686delT and crossed it with an elastase-Cre mouse to derive a bitransgenic mouse line with pancreas-specific over-expression of CEL carrying this disease-associated mutation (TgCEL). Following confirmation of murine pancreatic expression of the human transgene by real-time quantitative PCR, we phenotyped the mouse model fed a normal chow and compared it with mice fed a 60% high fat diet (HFD) as well as the effects of short-term and long-term cerulein exposure. RESULTS: Pancreatic exocrine function was normal in TgCEL mice on normal chow as assessed by serum lipid and lipid-soluble vitamin levels, fecal elastase and fecal fat absorption, and the normoglycemic mice exhibited normal pancreatic morphology. On 60% HFD, the mice gained weight to the same extent as controls, had normal pancreatic exocrine function and comparable glucose tolerance even after resuming normal diet and follow up up to 22 months of age. The cerulein-exposed TgCEL mice gained weight and remained glucose tolerant, and there were no detectable mutation-specific differences in serum amylase, islet hormones or the extent of pancreatic tissue inflammation. CONCLUSIONS: In this murine model of human CEL-MODY diabetes, we did not detect mutation-specific endocrine or exocrine pancreatic phenotypes, in response to altered diets or exposure to cerulein.

Large-scale proteomic approaches have identified numerous mitochondrial acetylated proteins; however in most cases, their regulation by acetyltransferases and deacetylases remains unclear. Sirtuin 3 (SIRT3) is an NAD(+)-dependent mitochondrial protein deacetylase that has been shown to regulate a limited number of enzymes in key metabolic pathways. Here, we use a rigorous label-free quantitative MS approach (called MS1 Filtering) to analyze changes in lysine acetylation from mouse liver mitochondria in the absence of SIRT3. Among 483 proteins, a total of 2,187 unique sites of lysine acetylation were identified after affinity enrichment. MS1 Filtering revealed that lysine acetylation of 283 sites in 136 proteins was significantly increased in the absence of SIRT3 (at least twofold). A subset of these sites was independently validated using selected reaction monitoring MS. These data show that SIRT3 regulates acetylation on multiple proteins, often at multiple sites, across several metabolic pathways including fatty acid oxidation, ketogenesis, amino acid catabolism, and the urea and tricarboxylic acid cycles, as well as mitochondrial regulatory proteins. The widespread modification of key metabolic pathways greatly expands the number of known substrates and sites that are targeted by SIRT3 and establishes SIRT3 as a global regulator of mitochondrial protein acetylation with the capability of coordinating cellular responses to nutrient status and energy homeostasis.

IGF-I is considered a primary inhibitor of GH secretion. Insulin may also play an important role in regulating GH levels because insulin, like IGF-I, can suppress GH synthesis and release in primary pituitary cell cultures and insulin is negatively correlated with GH levels in vivo. However, understanding the relative contribution insulin and IGF-I exert on controlling GH secretion has been hampered by the fact that circulating insulin and IGF-I are regulated in parallel and insulin (INSR) and IGF-I (IGFIR) receptors are structurally/functionally related and ubiquitously expressed. To evaluate the separate roles of insulin and IGF-I in directly regulating GH secretion, we used the Cre/loxP system to knock down the INSR and IGFIR in primary mouse pituitary cell cultures and found insulin-mediated suppression of GH is independent of the IGFIR. In addition, pharmacological blockade of intracellular signals in both mouse and baboon cultures revealed insulin requires different pathways from IGF-I to exert a maximal inhibitory effect on GH expression/release. In vivo, somatotrope-specific knockout of INSR (SIRKO) or IGFIR (SIGFRKO) increased GH levels. However, comparison of the pattern of GH release, GH expression, somatotrope morphometry, and pituitary explant sensitivity to acute GHRH challenge in lean SIRKO and SIGFRKO mice strongly suggests the primary role of insulin in vivo is to suppress GH release, whereas IGF-I serves to regulate GH synthesis. Finally, SIRKO and/or SIGFRKO could not prevent high-fat, diet-induced suppression of pituitary GH expression, indicating other factors/tissues are involved in the decline of GH observed with weight gain.

Members of the acyl-CoA thioesterase (Acot) gene family hydrolyze fatty acyl-CoAs, but their biological functions remain incompletely understood. Thioesterase superfamily member 2 (Them2; synonym Acot13) is enriched in oxidative tissues, associated with mitochondria, and relatively specific for long chain fatty acyl-CoA substrates. Using Them2(-/-) mice, we have demonstrated key roles for Them2 in regulating hepatic glucose and lipid metabolism. However, reduced body weights and decreased adiposity in Them2(-/-) mice observed despite increased food consumption were not well explained. To explore a role in thermogenesis, mice were exposed to ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C). In response to short term (24-h) exposures to decreasing ambient temperatures, Them2(-/-) mice exhibited increased adaptive responses in physical activity, food consumption, and energy expenditure when compared with Them2(+/+) mice. By contrast, genotype-dependent differences were not observed in mice that were equilibrated (96 h) at each ambient temperature. In brown adipose tissue, the absence of Them2 was associated with reduced lipid droplets, alterations in the ultrastructure of mitochondria, and increased expression of thermogenic genes. Indicative of a direct regulatory role for Them2 in heat production, cultured primary brown adipocytes from Them2(-/-) mice exhibited increased norepinephrine-mediated triglyceride hydrolysis and increased rates of O2 consumption, together with elevated expression of thermogenic genes. At least in part by regulating intracellular fatty acid channeling, Them2 functions in brown adipose tissue to suppress adaptive increases in energy expenditure.

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.

Visceral and s.c. fat exhibit different intrinsic properties, including rates of lipolysis, and are associated with differential risk for the development of type 2 diabetes. These effects are in part related to cell autonomous differences in gene expression. In the present study, we show that expression of Shox2 (Short stature homeobox 2) is higher in s.c. than visceral fat in both rodents and humans and that levels are further increased in humans with visceral obesity. Fat-specific disruption of Shox2 in male mice results in protection from high fat diet-induced obesity, with a preferential loss of s.c. fat. The reduced adipocyte size is secondary to a twofold increase in the expression of β3 adrenergic receptor (Adrb3) at both the mRNA and protein level and a parallel increase in lipolytic rate. These effects are mimicked by knockdown of Shox2 in C3H10T1/2 cells. Conversely, overexpression of Shox2 leads to a repression of Adrb3 expression and decrease lipolytic rate. Shox2 does not affect differentiation but directly interacts with CCAAT/enhancer binding protein alpha and attenuates its transcriptional activity of the Adrb3 promoter. Thus, Shox2 can regulate the expression of Adrb3 and control the rate of lipolysis and, in this way, exerts control of the phenotypic differences between visceral and s.c. adipocytes.

The sterol sensor SCAP is a key regulator of SREBP-2, the major transcription factor controlling cholesterol synthesis. Recently, we showed that there is a global down-regulation of cholesterol synthetic genes, as well as SREBP-2, in the brains of diabetic mice, leading to a reduction of cholesterol synthesis. We now show that in mouse models of type 1 and type 2 diabetes, this is, in part, the result of a decrease of SCAP. Homozygous disruption of the Scap gene in the brains of mice causes perinatal lethality associated with microcephaly and gliosis. Mice with haploinsufficiency of Scap in the brain show a 60% reduction of SCAP protein and ~30% reduction in brain cholesterol synthesis, similar to what is observed in diabetic mice. This results in impaired synaptic transmission, as measured by decreased paired pulse facilitation and long-term potentiation, and is associated with behavioral and cognitive changes. Thus, reduction of SCAP and the consequent suppression of cholesterol synthesis in the brain may play an important role in the increased rates of cognitive decline and Alzheimer disease observed in diabetic states.