The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of γ-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type γ-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the γ-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.

Catch-up growth, a risk factor for later obesity, type 2 diabetes, and cardiovascular diseases, is characterized by hyperinsulinemia and an accelerated rate for recovering fat mass, i.e., catch-up fat. To identify potential mechanisms in the link between hyperinsulinemia and catch-up fat during catch-up growth, we studied the in vivo action of insulin on glucose utilization in skeletal muscle and adipose tissue in a previously described rat model of weight recovery exhibiting catch-up fat caused by suppressed thermogenesis per se. To do this, we used euglycemic-hyperinsulinemic clamps associated with the labeled 2-deoxy-glucose technique. After 1 week of isocaloric refeeding, when body fat, circulating free fatty acids, or intramyocellular lipids in refed animals had not yet exceeded those of controls, insulin-stimulated glucose utilization in refed animals was lower in skeletal muscles (by 20 -43%) but higher in white adipose tissues (by two-to threefold). Furthermore, fatty acid synthase activity was higher in adipose tissues from refed animals than from fed controls. These results suggest that suppressed thermogenesis for the purpose of sparing glucose for catch-up fat, via the coordinated induction of skeletal muscle insulin resistance and adipose tissue insulin hyperresponsiveness, might be a central event in the link between catch-up growth, hyperinsulinemia and risks for later metabolic syndrome.

We report here studies that integrate data of respiration rate from mouse skeletal muscle in response to leptin and pharmacological interference with intermediary metabolism, together with assays for phosphatidylinositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK). Our results suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle is mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that this cycle requires both PI3K and AMPK signaling. This substrate cycling linking glucose and lipid metabolism to thermogenesis provides a novel thermogenic mechanism by which leptin protects skeletal muscle from excessive fat storage and lipotoxicity. Keywords: Obesity; Diabetes; Lipotoxicity; Gluco-lipotoxicity; Insulin resistance; Phosphatidylinositol 3-kinase; AMP-activated protein kinase; Sterol regulatory element binding protein-1c IntroductionSkeletal muscle, which accounts for 30-40% of body mass in mammals, is an important site for glucose disposal, lipid oxidation and thermogenesis whose impairments contribute to the pathogenesis of obesity and type 2 diabetes. It has long been suspected that these metabolic events are often interdependent in normal and disease states [1,2], but a mechanistic link between glucose and lipid metabolism to skeletal muscle thermogenesis is still ill-defined. Leptin, an adipocyte-derived hormone which is well known for its role in weight regulation, has also been shown to protect insulin-sensitive tissues like skeletal muscle against excessive fat storage that can lead to functional impairments known as lipotoxicity [3]. The demonstrations that leptin can act directly on skeletal muscle, specifically via the long form of the leptin receptor (ObRb), to stimulate glucose utilization [4], lipid oxidation through AMPactivated protein kinase (AMPK) [5,6] or thermogenesis in a phosphatidylinositol 3-kinase (PI3K)-dependent manner [7], have provided the impetus to investigate the mechanisms by which muscle substrate metabolism and thermogenesis are interdependent. Although the mechanisms leading to increased fatty acid oxidation in skeletal muscle in response to leptin have been described in molecular details [6], those underlying its effects on thermogenesis are still unknown, amid continuing controversies concerning the role of novel uncoupling proteins, UCP2 and UCP3, as effectors of skeletal muscle thermogenesis [3,8,9]. Furthermore, the mechanism by which glucose and lipid metabolism are linked to thermogenesis in response to leptin's direct effect on skeletal muscle is unknown. With the objective of elucidating the mechanisms by which leptin exerts its direct effect on skeletal muscle thermogenesis, we report here a study that integrates data of respiration rate from intact mouse skeletal muscle ex vivo in response to leptin and pharmacological interference with key control points of intermediary metabolism, together with biochemical measurements for PI3K and AMPK signaling. Materials and methods Mice and ...

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.