The objective is to investigate the role of insulin-like growth factor 1 (IGF-1) in the regulation of core body temperature. Sequencing cDNA libraries from individual warm-sensitive neurons from the preoptic area (POA) of the hypothalamus, a region involved in the central control of thermoregulation, identified neurons that express both IGF-1 receptor (IGF-1R) and insulin receptor transcripts. The effects of administration of IGF-1 into the POA was measured by radiotelemetry monitoring of core temperature, brown adipose tissue (BAT) temperature, metabolic assessment, and imaging of BAT by positron emission tomography of 2-[(18)F]fluoro-2-deoxyglucose uptake combined with computed tomography. IGF-1 injection into the POA caused dose-dependent hyperthermia that could be blocked by pretreatment with the IGF-1R tyrosine kinase inhibitor, PQ401. The IGF-1-evoked hyperthermia involved activation of brown adipose tissue and was accompanied by a switch from glycolysis to fatty acid oxidation as a source of energy as shown by lowered respiratory exchange ratio. Transgenic mice that lack neuronal insulin receptor expression in the brain (NIRKO mice) were unable to mount the full hyperthermic response to IGF-1, suggesting that the IGF-1 mediated hyperthermia is partly dependent on expression of functional neuronal insulin receptors. These data indicate a novel thermoregulatory role for both IGF-1R and neuronal insulin receptors in IGF-1 activation of BAT and hyperthermia. These central effects of IGF-1 signaling may play a role in regulation of metabolic rate, aging, and the risk of developing type 2 diabetes.

Increased intraabdominal (visceral) fat is associated with a high risk of diabetes and metabolic syndrome. We have previously shown that the mesodermal developmental transcription factor Tbx15 is highly differentially expressed between visceral and subcutaneous (s.c.) fat in both humans and rodents, and in humans visceral fat Tbx15 expression is decreased in obesity. Here we show that, in mice, Tbx15 is 260-fold more highly expressed in s.c. preadipocytes than in epididymal preadipocytes. Overexpression of Tbx15 in 3T3-L1 preadipocytes impairs adipocyte differentiation and decreases triglyceride content. This defect in differentiation can be corrected by stimulating cells with the PPARγ agonist rosiglitazone (Rosi). However, triglyceride accumulation remains decreased by ∼50%, due to a decrease in basal lipogenic rate and increase in basal lipolytic rate. 3T3-L1 preadipocytes overexpressing Tbx15 also have a 15% reduction in mitochondrial mass and a 28% reduction in basal mitochondrial respiration (P = 0.004) and ATP turnover (P = 0.02), and a 45% (P = 0.003) reduction in mitochondrial respiratory capacity. Thus, differential expression of Tbx15 between fat depots plays an important role in the interdepot differences in adipocyte differentiation, triglyceride accumulation, and mitochondrial function that may contribute to the risk of diabetes and metabolic disease.

The endoplasmic reticulum (ER) consists of an interconnected, membranous network that is the major site for the synthesis and folding of integral membrane and secretory proteins. Within the ER lumen, protein folding is facilitated by molecular chaperones and a variety of enzymes that ensure that polypeptides obtain their appropriate, tertiary conformation (Dobson, C. M. (2004). Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol. 15, 3-16; Ni, M., and Lee, A. S. (2007). ER chaperones in mammalian development and human diseases. FEBS Lett. 581, 3641-3651.). Physiological conditions that increase protein synthesis or stimuli that disturb the processes by which proteins obtain their native conformation, create an imbalance between the protein-folding demand and capacity of the ER. This results in the accumulation of unfolded or improperly folded proteins in the ER lumen and a state of ER stress. The cellular response, referred to as the unfolded protein response (UPR), results in activation of three linked signal transduction pathways: PKR-like kinase (PERK), inositol requiring 1 α (IRE1α), and activating transcription factor 6α (ATF6α) (Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell. Biol. 8, 519-529; Schroder, M., and Kaufman, R. (2005). ER stress and the unfolded protein response. Mutat. Res./Fundam. Mol. Mech. Mutagen. 569, 29-63.). Collectively, the combined actions of these signaling cascades serve to reduce ER stress through attenuation of translation to reduce protein synthesis and through activation of transcriptional programs that ultimately serve to increase ER protein-folding capacity. Recently, we and Park et al. have characterized a novel function for the p85α and p85β subunits as modulators of the UPR by virtue of their ability to facilitate the nuclear entry of XBP-1s following induction of ER stress (Park, S. W., Zhou, Y., Lee, J., Lu, A., Sun, C., Chung, J., Ueki, K., and Ozcan, U. (2010). Regulatory subunits of PI3K, p85alpha and p85 beta, interact with XBP1 and increase its nuclear translocation. Nat. Med. 16, 429-437; Winnay, J. N., Boucher, J., Mori, M. A., Ueki, K., and Kahn, C. R. (2010). A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response. Nat. Med. 16, 438-445.). This chapter describes the recently elucidated role for the regulatory subunits of PI 3-kinase as modulators of the UPR and provides methods to measure UPR pathway activation.

Acetylation has recently emerged as an important mechanism for controlling a broad array of proteins mediating cellular adaptation to metabolic fuels. Acetylation is governed, in part, by SIRTs (sirtuins), class III NAD(+)-dependent deacetylases that regulate lipid and glucose metabolism in liver during fasting and aging. However, the role of acetylation or SIRTs in pathogenic hepatic fuel metabolism under nutrient excess is unknown. In the present study, we isolated acetylated proteins from total liver proteome and observed 193 preferentially acetylated proteins in mice fed on an HFD (high-fat diet) compared with controls, including 11 proteins not previously identified in acetylation studies. Exposure to the HFD led to hyperacetylation of proteins involved in gluconeogenesis, mitochondrial oxidative metabolism, methionine metabolism, liver injury and the ER (endoplasmic reticulum) stress response. Livers of mice fed on the HFD had reduced SIRT3 activity, a 3-fold decrease in hepatic NAD(+) levels and increased mitochondrial protein oxidation. In contrast, neither SIRT1 nor histone acetyltransferase activities were altered, implicating SIRT3 as a dominant factor contributing to the observed phenotype. In Sirt3⁻(/)⁻ mice, exposure to the HFD further increased the acetylation status of liver proteins and reduced the activity of respiratory complexes III and IV. This is the first study to identify acetylation patterns in liver proteins of HFD-fed mice. Our results suggest that SIRT3 is an integral regulator of mitochondrial function and its depletion results in hyperacetylation of critical mitochondrial proteins that protect against hepatic lipotoxicity under conditions of nutrient excess.