Functionalization of C-H bonds in acetophenone oximes with arylacetic acids and elemental sulfur.

Fused thieno[3,2-d]thiazoles were synthesized via a coupling of acetophenone ketoximes, arylacetic acids, and elemental sulfur in the presence of Li2CO3 base. Functionalities including chloro, bromo, fluoro, trifluoromethyl, and pyridyl groups were compatible with reaction conditions. High yields and excellent regioselectivities were obtained even if meta-substituted ketoxime acetates were used. Ethyl esters of heteroarylacetic acids were competent substrates, which is very rare in the literature. Our method would offer a convenient protocol to afford polyheterocyclic structures from simple substrates.

Late Pseudoaneurysm After Access Site Closure with Manta in Transfemoral Aortic Valve Implantation.

INTRODUCTION: Access site vascular complications in transfemoral transcatheter aortic valve implantation (TF-TAVI) are still a major concern. Recently, a novel collagen plug based closure device (Manta) was introduced. The results from the first reports on Manta are very promising, but not much is known about the long-term patency. REPORT: A case of late pseudoaneurysm after access site arterial closure with Manta in TF-TAVI is described. The patient presented five weeks after left sided TF-TAVI with pain and claudication like symptoms in the left leg. CT angiography revealed a pseudoaneurysm at the puncture site. The patient was successfully treated by vascular surgery. DISCUSSION: The results from recent peri-operative reports on the Manta vascular closure device (VCD) are promising, but not much is known about the long-term patency. In the present report a patient is described who developed a pseudoaneurysm several weeks after access site closure with Manta. To the authors' knowledge, no such late access site complications after use of the Manta VCD have been reported previously.

PKCδ regulates hepatic insulin sensitivity and hepatosteatosis in mice and humans.

C57BL/6J and 129S6/Sv (B6 and 129) mice differ dramatically in their susceptibility to developing diabetes in response to diet- or genetically induced insulin resistance. A major locus contributing to this difference has been mapped to a region on mouse chromosome 14 that contains the gene encoding PKCδ. Here, we found that PKCδ expression in liver was 2-fold higher in B6 versus 129 mice from birth and was further increased in B6 but not 129 mice in response to a high-fat diet. PRKCD gene expression was also elevated in obese humans and was positively correlated with fasting glucose and circulating triglycerides. Mice with global or liver-specific inactivation of the Prkcd gene displayed increased hepatic insulin signaling and reduced expression of gluconeogenic and lipogenic enzymes. This resulted in increased insulin-induced suppression of hepatic gluconeogenesis, improved glucose tolerance, and reduced hepatosteatosis with aging. Conversely, mice with liver-specific overexpression of PKCδ developed hepatic insulin resistance characterized by decreased insulin signaling, enhanced lipogenic gene expression, and hepatosteatosis. Therefore, changes in the expression and regulation of PKCδ between strains of mice and in obese humans play an important role in the genetic risk of hepatic insulin resistance, glucose intolerance, and hepatosteatosis; and thus PKCδ may be a potential target in the treatment of metabolic syndrome.

Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat.

Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1(+)/CD45(-)/Mac1(-); referred to as Sca-1(+) progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1(+) cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development.

Specific roles of the p110alpha isoform of phosphatidylinsositol 3-kinase in hepatic insulin signaling and metabolic regulation.

The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism.

Transplantation of adipose tissue and stem cells: role in metabolism and disease.

Humans and other mammals have three main adipose tissue depots: visceral white adipose tissue, subcutaneous white adipose tissue and brown adipose tissue, each of which possesses unique cell-autonomous properties. In contrast to visceral adipose tissue, which can induce detrimental metabolic effects, subcutaneous white adipose tissue and brown adipose tissue have the potential to benefit metabolism by improving glucose homeostasis and increasing energy consumption. In addition, adipose tissue contains adipose-derived stem cells, which possess the ability to differentiate into multiple lineages, a property that might be of value for the repair or replacement of various damaged cell types. Adipose tissue transplantation has primarily been used as a tool to study physiology and for human reconstructive surgery. Transplantation of adipose tissue is, however, now being explored as a possible tool to promote the beneficial metabolic effects of subcutaneous white adipose tissue and brown adipose tissue, as well as adipose-derived stem cells. Ultimately, the clinical applicability of adipose tissue transplantation for the treatment of obesity and metabolic disorders will reside in the achievable level of safety, reliability and efficacy compared with other treatments.

Adipose depots possess unique developmental gene signatures.

We have previously demonstrated that subcutaneous and intra-abdominal adipose tissue show different patterns of expression for developmental genes (Shox2, En1, Tbx15 Hoxa5, Hoxc8, and Hoxc9), and that the expression level of Tbx15 and Hoxa5 in humans correlated with the level of obesity and fat distribution. To further explore the role of these developmental genes in adipose tissue, we have characterized their expression in different adipose depots in mice, and studied their regulation in obesity and by fasting. Developmental and adipogenic gene expression was compared in two subcutaneous and three intra-abdominal white adipose tissue (WAT) depots as well as brown adipose tissue (BAT) from lean or obese mice in a fed or fasting state. Each of these six adipose depots display a unique pattern of developmental gene expression, whereas expression of adipogenic transcription factors PPARgamma2 C/EBPalpha, beta, and Delta showed constant expression levels in all depots. Expression levels of developmental genes were similar in obese (ob/ob and high-fat diet (HFD)) and lean mice in most depots. Fasting systematically decreased expression of Hoxc8, PPARgamma2, and increased C/EBPDelta in both lean and ob/ob mice, but produced only variable changes in the expression of other developmental and adipogenic genes. These data indicate that each fat depot has a unique developmental gene expression signature, which is largely independent of nutritional state. This finding further supports a fundamental role of developmental genes in fat distribution and the development and/or function of specific adipose tissue depots.

Sex and depot differences in adipocyte insulin sensitivity and glucose metabolism.

OBJECTIVE: To investigate how insulin sensitivity and glucose metabolism differ in adipocytes between different fat depots of male and female mice and how sex steroids contribute to these differences. RESEARCH DESIGN AND METHODS: Adipocytes from intra-abdominal/perigonadal (PG) and subcutaneous (SC) adipose tissue from normal, castrated, or steroid-implanted animals were isolated and analyzed for differences in insulin sensitivity and glucose metabolism. RESULTS: Adipocytes from both PG and SC depots of females have increased lipogenic rates compared with those from males. In females, intra-abdominal PG adipocytes are more insulin-sensitive than SC adipocytes and more insulin-sensitive than male adipocytes from either depot. When stimulated by low physiological concentrations of insulin, female PG adipocytes show a robust increase in Akt and extracellular signal-related kinase (ERK) phosphorylation and lipogenesis, whereas male adipocytes show activation only at higher insulin concentrations. Adipocytes from females have higher mRNA/protein levels of several genes involved in glucose and lipid metabolism. After castration, adipocytes of male mice showed increased insulin sensitivity and increased lipogenic rates, whereas adipocytes of females demonstrate decreased lipid production. Increasing estrogen above physiological levels, however, also reduced lipid synthesis in females, whereas increasing dihydrotestosterone in males had no effect. CONCLUSIONS: There are major sex differences in insulin sensitivity in adipose tissue, particularly in the intra-abdominal depot, that are regulated by physiological levels of sex steroids. The increased sensitivity to insulin and lipogenesis observed in adipocytes from females may account for their lower level of insulin resistance and diabetes risk despite similar or higher fat content than in males.

Beneficial effects of subcutaneous fat transplantation on metabolism.

Subcutaneous (SC) and visceral (VIS) obesity are associated with different risks of diabetes and the metabolic syndrome. To elucidate whether these differences are due to anatomic location or intrinsic differences in adipose depots, we characterized mice after transplantation of SC or VIS fat from donor mice into either SC or VIS regions of recipient mice. The group with SC fat transplanted into the VIS cavity exhibited decreased body weight, total fat mass, and glucose and insulin levels. These mice also exhibited improved insulin sensitivity during hyperinsulinemic-euglycemic clamps with increased whole-body glucose uptake, glucose uptake into endogenous fat, and insulin suppression of hepatic glucose production. These effects were observed to a lesser extent with SC fat transplanted to the SC area, whereas VIS fat transplanted to the VIS area was without effect. These data suggest that SC fat is intrinsically different from VIS fat and produces substances that can act systemically to improve glucose metabolism.

Skeletal muscle-selective knockout of LKB1 increases insulin sensitivity, improves glucose homeostasis, and decreases TRB3.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a > 80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.

Phosphoinositide 3-kinase regulatory subunit p85alpha suppresses insulin action via positive regulation of PTEN.

The phosphoinositide 3-kinase (PI3K) pathway is central to the metabolic actions of insulin on liver. Here, we show that mice with a liver-specific deletion of the p85alpha regulatory subunit of PI3K (L-Pik3r1KO) exhibit a paradoxical improvement of hepatic and peripheral insulin sensitivity. Although PI3K enzymatic activity is diminished in L-Pik3r1KO livers because of a reduced level of regulatory and catalytic subunits of PI3K, insulin-stimulated Akt activity is actually increased. This increased Akt activity correlates with increased phosphatidylinositol (3,4,5)-trisphosphate levels which are due, at least in part, to diminished activity of the (3,4,5)-trisphosphate phosphatase PTEN. Thus, the regulatory subunit p85alpha is a critical modulator of insulin sensitivity in vivo not only because of its effects on PI3K activation, but also as a regulator of PTEN activity.

Hyperinsulinemia, but not other factors associated with insulin resistance, acutely enhances colorectal epithelial proliferation in vivo.

The similarity in risk factors for insulin resistance and colorectal cancer (CRC) led to the hypothesis that markers of insulin resistance, such as elevated circulating levels of insulin, glucose, fatty acids, and triglycerides, are energy sources and growth factors in the development of CRC. The objective was thus to examine the individual and combined effects of these circulating factors on colorectal epithelial proliferation in vivo. Rats were fasted overnight, randomized to six groups, infused iv with insulin, glucose, and/or Intralipid for 10 h, and assessed for 5-bromo-2-deoxyuridine labeling of replicating DNA in colorectal epithelial cells. Intravenous infusion of insulin, during a 10-h euglycemic clamp, increased colorectal epithelial proliferation in a dose-dependent manner. The addition of hyperglycemia to hyperinsulinemia did not further increase proliferation. Intralipid infusion alone did not affect proliferation; however, the combination of insulin, glucose, and Intralipid infusion resulted in greater hyperinsulinemia than the infusion of insulin alone and further increased proliferation. Insulin infusion during a 10-h euglycemic clamp decreased total IGF-I levels and did not affect insulin sensitivity. These results provide evidence for an acute role of insulin, at levels observed in insulin resistance, in the proliferation of colorectal epithelial cells in vivo.

Direct measure of insulin sensitivity with the hyperinsulinemic-euglycemic clamp and surrogate measures of insulin sensitivity with the oral glucose tolerance test: correlations with aberrant crypt foci promotion in rats.

The similarity in lifestyle risk factors for the development of colorectal cancer (CRC) and type 2 diabetes suggests that there are common underlying pathogenic mechanisms. High-risk lifestyle factors may lead to insulin resistance that, through increased circulating levels of energy substrates, insulin, and insulin-like growth factor-1, may promote the development of CRC. The objective was to determine the extent to which direct and surrogate measures of insulin resistance correlate with multiplicity of aberrant crypt foci, which are putative precursors of CRC. Rats were initiated with the carcinogen azoxymethane, then fed low, intermediate, or high saturated fat diets. Metabolic parameters were assessed at 50 days and ACF at 100 days after initiation. Results indicate that CRC promotion was most strongly correlated with direct measures of insulin sensitivity as assessed with the hyperinsulinemic-euglycemic clamp (r = -0.52, P < 0.009). Practical surrogate measures of insulin resistance such as insulin levels at 180 min after an oral glucose load were strongly correlated with direct measures of insulin sensitivity (r = -0.61, P < 0.001) and with CRC promotion (r = 0.42, P = 0.044) in this animal model. Fasting levels of glucose, insulin, total insulin-like growth factor-1, nonesterified fatty acids, and triglyceride, as well as body weight and insulin sensitivity indices (such as fasting insulin resistance index, quantitative insulin sensitivity check index, homeostasis model assessment formula, insulin sensitivity index of glycemia, oral glucose insulin sensitivity, and composite insulin sensitivity index for the hepatic and peripheral tissues) were all less strongly correlated with direct measures of insulin sensitivity and all poorly correlated with CRC promotion in this animal model. These correlations do not prove causality, however, they suggest possible mechanisms linking diet, insulin resistance with its related parameters, and promotion of CRC.