BACKGROUND
insulin resistance is a metabolic condition found relatively frequently among humans with chronic hyperinsulinemia and in experimental animal models with defective insulin signaling  <cit> .
recently, a link has been established between peripheral insulin sensitivity and the retinol  metabolism, and insulin resistance in rodents and humans has been linked to abnormalities of the vitamin a signaling pathway  <cit> .
according to these studies, impaired glucose uptake in adipose tissue results in secondary systemic insulin resistance through release of the adipose-derived serum rbp <dig>  <cit> .
however, it is unknown whether rbp <dig> effects on insulin sensitivity are vitamin a-dependent or vitamin a-independent.
rbp <dig>  is mainly produced by the liver but also by adipocytes  <cit> .
in plasma, retinol-rbp <dig> is found in an equimolar complex with transthyretin , which is a thyroid hormone transport protein that is synthesized in and secreted from the liver.
this ternary complex prevents retinol-rbp <dig> excretion by the kidney  <cit> .
by impairing insulin signaling in muscle, rbp <dig> inhibits glucose uptake and interferes with insulin-mediated suppression of glucose production in the liver, causing blood glucose levels to rise  <cit> .
conversely, mice lacking the rbp <dig> gene show increased insulin sensitivity, and normalizing increased rbp <dig> serum levels improves insulin resistance and glucose intolerance  <cit> .
hmga <dig> is a small basic protein that binds to adenine-thymine  rich regions of dna and functions mainly as a specific cofactor for gene activation  <cit> .
hmga <dig> by itself has no intrinsic transcriptional activity; rather, it can transactivate promoters through mechanisms that facilitate the assembly and stability of a multicomponent enhancer complex, the so-called enhanceosome, that drives gene transcription  <cit> .
as part of an investigation into the molecular basis regulating the human insulin receptor gene, we previously showed that hmga <dig> is required for proper insulin receptor gene transcription  <cit> .
more recently, we showed that loss of hmga <dig> expression, induced in mice by disrupting the hmga <dig> gene, caused a type 2-like diabetic phenotype, in which, however, impaired glucose tolerance and overt diabetes coexisted with a condition of peripheral insulin hypersensitivity  <cit> .
concomitant insulin resistance and insulin hypersensitivity in peripheral tissues may paradoxically coexist as observed in livers of lipodystrophic and ob/ob mice  <cit> , as well as in cdk <dig> knockout mice with defective pancreatic beta cell development and blunted insulin secretion  <cit> .
the hypothesis that the paradoxical insulin hypersensitivity of hmga1-deficient mice could be due to a deficit, in these animals, of rbp <dig> is supported by our data.
herein, by employing the hmga1-knockout mouse model, we provide compelling evidence for the identification of a novel biochemical pathway involving hmga <dig> and rbp <dig>  whose activation by the camp pathway may play an important role in maintaining glucose metabolism homeostasis in vivo, in both rodents and humans.
the importance of hmga <dig> in rbp <dig> gene transcription was substantiated in hmga1-deficient mice, in which loss of hmga <dig> expression considerably decreased rbp <dig> mrna abundance and rbp <dig> protein production.
RESULTS
rbp <dig> gene transcription is induced by hmga <dig> and camp
we first performed experiments to see whether hmga <dig> had a role in activating the mouse rbp <dig> gene promoter at the transcriptional level.
to test this possibility, hepg <dig> human hepatoma cells and mouse hepa <dig> hepatoma cells were cotransfected transiently with mouse rbp4-luc reporter plasmid plus increasing amounts of the hmga <dig> expression vector.
as shown in figure  <dig>  overexpression of hmga <dig> considerably increased rbp4-luc activity in both cell types and this effect occurred in a dose-dependent manner.
consistent with these results, rbp <dig> mrna abundance was increased in cells overexpressing hmga <dig> and was reduced in cells pretreated with sirna targeting hmga <dig> , indicating that activation of the rbp <dig> gene requires hmga <dig>  these data were substantiated by chromatin immunoprecipitation  assay, showing that binding of hmga <dig> to the endogenous rbp <dig> locus was increased in whole, intact hepg <dig> and hepa <dig> cells naturally expressing hmga <dig>  and was decreased in cells exposed to sirna against hmga <dig> .
based on these results, in addition to previous observations indicating that camp, or agents which elevate intracellular camp, increase rbp transcript levels  <cit> , we were interested to see whether a functional link could be established between camp, hmga <dig>  and rbp <dig>  to this end, we first confirmed and extended the observation made by jessen and satre  <cit>  that rbp is induced by camp in hepa <dig> cells.
as measured by northern blot analysis of total rna , rbp <dig> mrna increased ≈ 5-fold over the basal level in hepa <dig> cells treated with  <dig>  mm 8-bromo camp , a standard concentration for c-amp induction experiments  <cit> .
as shown in figure  <dig>  rbp <dig> mrna levels increased starting at  <dig> h, peaking at  <dig> h and then declining, suggesting a transient transcriptional stimulation.
to establish whether hmga <dig> was required for basal and camp-dependent rbp <dig> transcription, we transfected the hmga <dig> expression vector in hepa <dig> cells treated or not with br-camp and rbp <dig> protein levels were analyzed  <dig> h later by western blot.
as shown in figure  <dig>  rbp <dig> protein expression was enhanced in cells overexpressing hmga <dig> and even further in cells treated with camp, in which an increase in hmga <dig> protein expression was simultaneously observed, suggesting that induction of rbp <dig> by camp may occur, at least in part, through activation of endogenous hmga <dig> expression.
this hypothesis was supported by the fact that rbp <dig> was reduced in camp treated cells in which endogenous levels of hmga <dig> were specifically lowered by transfecting cells with hmga <dig> antisense expression plasmid .
however, further experiments are needed to fully explain the role of camp on hmga <dig> expression.
the functional significance of hmga <dig> in rbp <dig> gene expression was confirmed in transient transcription assays in hepa <dig>  cells, in which overexpression of hmga <dig> caused an increase in both basal and camp-induced luc activity from the mouse rbp-luc reporter plasmid .
this effect was substantiated in hek- <dig> camp-responsive cells, a cell line ideally suited for studying the effects of hmga <dig> on transcription since it does not express appreciable levels of this protein.
as shown in figure  <dig>  in support of the role that hmga <dig> plays in the context of rbp <dig> gene, the direct effect of camp was less effective in promoting rbp <dig> transcription in hek- <dig> cells expressing low levels of hmga <dig>  becoming considerably higher in cells with forced expression of hmga <dig>
thus, these data together demonstrate that hmga <dig> is of major importance for transcriptional regulation of the rbp <dig> gene, and indicate that a functional link exists between camp, hmga <dig>  and rbp <dig>
hmga1-deficient mice have reduced expression of rbp <dig> in liver and fat tissue and reduced serum rbp <dig> levels
in the light of the above experimental results, indicating that hmga <dig> plays a positive role in rbp <dig> gene transcription in living cultured cells, it was interesting to analyze the functional consequences of genetic ablation of hmga <dig> on rbp <dig> in vivo, in hmga1-knockout mice.
to this end, we performed studies aimed at investigating the expression of rbp <dig> mrna and protein in hmga1-deficient mice and wild-type controls.
as shown in figure  <dig>  rbp <dig> mrna was severely attenuated in both liver and fat from hmga1-null mice, and reduced by 50% in hmga <dig> heterozygous mutants, as assessed by real-time quantitative polymerase chain reaction .
reduced rbp <dig> mrna levels in liver and adipose tissue paralleled the decrease in rbp <dig> serum levels as detected by western blot analysis of serum samples from age- and body weight-matched mice with diverse genotypes , thereby showing the requirement of hmga <dig> for full rbp <dig> expression in whole animals.
hmga <dig> and rbp <dig> expression increase in liver and fat of normal mice after intraperitoneal glucagon injection
based on our observations in intact cultured cells, indicating a role for the camp signaling pathway in hmga <dig> and rbp <dig> gene expression, camp-inducible transcriptional activation of the hmga <dig> and rbp <dig> genes was investigated in vivo, in whole animals, by systemic administration of the intracellular camp-elevating hormone glucagon.
under these conditions, glucagon-stimulated camp responses in terms of both hmga <dig> and rbp <dig> mrna expression were first analyzed in wild-type control mice.
consistent with our data in hepa <dig> cells, hmga <dig> and rbp <dig> mrna levels significantly increased in liver and fat of normal mice after intraperitoneal injection of glucagon .
time course analyses revealed that the induction and accumulation of hmga <dig> mrna preceded the expression of rbp <dig> mrna in both tissues.
in liver, rbp <dig> mrna appeared after that for hmga <dig>  peaked after  <dig> h following glucagon injection, and then remained at a plateau .
in fat, rbp <dig> mrna appeared at  <dig> h after hmga <dig> mrna, peaked after  <dig> h of glucagon stimulation, and decreased smoothly thereafter .
increased levels of hmga <dig> and rbp <dig> mrnas were paralleled by the increase of hmga <dig> and rbp <dig> protein expression, as measured by western blot analysis of proteins from liver and fat of glucagon-injected animals .
interestingly, when similar experiments were carried out in glucagon injected hmga1-deficient mice, tissue expression of rbp <dig> mrna was severely attenuated in liver and fat from heterozygous  and hmga1-null  animals , thereby indicating that hmga <dig> is indeed required for maximal induction of the rbp <dig> gene in vivo, in the whole organism, and that the glucagon/adenylate cyclase system regulates both hmga <dig> and rbp <dig> gene and protein expression.
consistent with this conclusion, liver rbp <dig> mrna and protein expression levels were lower in fed wild-type mice, becoming higher during fasting, when circulating glucagon increases .
as a measure of the glucagon efficacy in glucagon-injected mice, a liver biopsy was taken before and after glucagon injection, and camp levels in liver were determined for both control and hmga1-deficient mice .
no substantial difference was found in basal levels of camp .
after glucagon injection, hepatic levels of camp increased to  <dig>  μmol/g tissue in control mice, compared with  <dig>  and  <dig>  in hmga1-/- and hmga1+/- mice, respectively.
results similar to those shown in the inset of figure  <dig> were also obtained in epididymal and subcutaneous fat pads from control and mutant animals , thus indicating that the glucagon-stimulated camp synthesis did not differ among mice with diverse genotypes.
hmga1-deficient mice have increased glut <dig> expression and insulin signaling activity in skeletal muscle and fat
systemic insulin resistance has been associated with elevation of serum rbp <dig>  whereas genetic and pharmacological interventions aimed at decreasing serum rbp <dig> levels enhance insulin action and improve insulin sensitivity  <cit> .
increased peripheral insulin sensitivity during insulin-tolerance test was previously observed by us in hmga1-knockout mice  <cit> .
to verify whether a functional link indeed existed between hmga <dig> and rbp <dig>  and whether insulin hypersensitivity in hmga1-deficient mice could be mediated by the hmga1-rbp <dig> system, we carried out quantitative measurements of glut <dig> mrna transcript abundance.
examination by qrt-pcr showed a significant increase of glut <dig> transcripts in both skeletal muscle and adipose tissues from hmga1-deficient mice compared with controls .
accordingly, immunoblotting of muscle and fat tissue showed a 2- to 3-fold increase of glut <dig> in the insulin hypersensitive hmga1-knockout mice compared with controls , clearly indicating that an inverse correlation between rbp <dig> and glut <dig> indeed exists in vivo, in this animal model of diabetes, in which reduced rbp <dig> may contribute to the maintenance of glucose homeostasis by increasing insulin signaling and peripheral insulin sensitivity.
in agreement with this interpretation, the activation state of the protein kinase akt, an important downstream target of pi 3-kinase regulating insulin serum effects on glut <dig> translocation and carbohydrate metabolism  <cit> , was increased in mutant animals.
as shown in figure  <dig>  basal phospho-akt immunoreactivity was higher in skeletal muscle and adipose tissues from hmga1-deficient mice compared with wild-type controls, and this increase paralleled closely the increase of glut <dig> protein in adipose and muscle plasma membranes from heterozygous and homozygous hmga <dig> mutants.
in line with previous observations on transcriptional repression of the mouse glut <dig> gene by camp  <cit> , endocrine upregulation of glut <dig> in hmga1-deficient mice was substantiated further by in vitro experiments , indicating that in isolated adipocytes treated with br-camp, glut <dig> mrna was decreased in all three genotypes.
a positive correlation of rbp <dig> levels with markers of lipid metabolism adversely affecting insulin sensitivity has been reported recently in both clinical and experimental studies  <cit> .
hmga1-knockout mice had lower levels of serum free fatty acids , which might contribute to their improved insulin sensitivity.
recombinant rbp <dig> injection reduces glut <dig> and insulin signaling activity in muscle and fat tissue of hmga1-deficient mice, and attenuates insulin hypersensitivity of these animals
to demonstrate that increased insulin sensitivity in mutant mice was directly due to hmga <dig> regulation of rbp <dig>  we determined the effect of recombinant rbp <dig> administration on akt phosphorylation and glut <dig> protein expression in skeletal muscle from hmga1-deficient mice.
as shown in figure  <dig>  akt phosphorylation was reduced in muscle from rbp4-injected mutant animals compared with saline-injected hmga <dig> mutants.
the reduction in akt phosphorylation in these genotypes correlated inversely with rbp <dig> serum levels in the same animals  and paralleled the reduction of glut <dig> in skeletal muscle and adipose  plasma membranes , indicating that, in these conditions, activation of the akt-glut <dig> pathway is regulated, at least in part, by circulating rbp <dig>  plasma insulin levels were slightly higher in rbp4-injected mice, but no significant difference was found .
consistent with the condition of insulin hypersensitivity, we previously reported that the glucose-lowering effect of exogenous insulin was enhanced in hmga1-deficient mice during insulin-tolerance test   <cit> .
to support further the role of rbp <dig> in insulin hypersensitivity in hmga <dig> mutants, we have determined the effect of rbp <dig> administration on the glucose fall induced by insulin in these genotypes during itt.
as shown in figure  <dig>  injection of human rbp <dig> in heterozygous and homozygous hmga <dig> mutants caused a less dramatic fall in blood glucose levels, lessening the hypoglycemic response to intraperitoneal insulin observed in the saline-injected animals.
thus, taken together, our findings consistently support the role of hmga <dig> as a key element in the transcriptional regulation of genes involved in glucose metabolism and add new insights into the compensatory mechanisms that may contribute to counteract insulin resistance in vivo.
by directly regulating rbp <dig> gene transcription, hmga <dig> enhances peripheral insulin sensitivity, ensuring glucose uptake in skeletal muscle.
this, if on one hand might represent an adaptive mechanism to ameliorate insulin resistance in animals with a disadvantageous metabolic risk profile, on the other might indicate that the camp/hmga1-mediated rbp <dig> expression during fasting  may act physiologically to reduce insulin sensitivity in peripheral tissues, thereby contributing to the maintenance of euglycemia under this condition.
this was supported by the observation that after an overnight fasting period  plasma glucose concentration in wild-type mice was higher than that of hmga1-deficient mice .
discussion
we have previously shown that loss of hmga <dig> protein expression, induced in mice by disrupting the hmga <dig> gene, severely decreased insulin receptor expression  and phosphorylation in the major targets of insulin action, largely impaired insulin signaling, and reduced insulin secretion, producing a type 2-like diabetic phenotype in which defects in both peripheral insulin sensitivity and pancreatic beta-cell insulin secretion were coexpressed simultaneously  <cit> .
however, despite the severe decrease in insulin receptor signaling and insulin receptor production, the glucose-lowering effect of exogenous insulin was enhanced in hmga1-deficient mice during itt, and the glucose infusion rate necessary to maintain euglycemia was higher in mutant mice during hyperinsulinemic-euglycemic clamp  <cit> , supporting the existence of alternative pathways of insulin signaling promoting glucose uptake and disposal in certain adverse metabolic conditions such as those found in the hmga1-knockout mouse.
the existence of signaling pathways promoting glucose uptake and utilization in peripheral tissues through mechanisms that are independent of insulin has been postulated before, on the basis of experimental observations supporting the existence of molecular circuits/pathways that can compensate for the decrease in insulin-stimulated glucose uptake in vivo, in both animal models and human patients with type  <dig> diabetes  <cit> .
however, how these compensatory mechanisms are activated has remained hitherto largely undefined.
as previously shown, consistent with the ubiquitous distribution of hmga <dig>  insulin receptor expression was also reduced in pancreatic tissue from hmga1-deficient mice  <cit> .
loss of insulin secretion in response to glucose has been reported in irβ knockout mice with tissue-specific knockout of the insulin receptor in pancreatic beta cells  <cit> .
as in the irβ knockout mice, plasma insulin after glucose challenge was considerably reduced in hmga1-mutant animals, in which the acute first-phase insulin secretory response was severely blunted  <cit> , indicating a glucose-induced insulin secretory defect.
in addition, substantial abnormalities in pancreatic islet morphology and size have been described in hmga1-knockout mice  <cit> , indicating that decreased insulin secretion in this genotype may also depend on reduced beta-cell mass.
thus, defects in both pancreatic beta-cell insulin secretion and peripheral insulin action coexist simultaneously in this knockout mouse model of diabetes, in which activation of compensatory mechanisms to efficiently overcome these metabolic abnormalities may be of vital importance.
downregulation of glut <dig> in adipose tissue is a typical feature of insulin-resistant states, such as obesity and type  <dig> diabetes  <cit> .
it has been found that the decrease in glut <dig> expression that occurs in the fatty tissue of obese animals and humans is accompanied by increased expression and secretion of the adipocyte-derived rbp <dig> fraction  <cit> , suggesting that rbp <dig> production is tightly regulated by adipose tissue glucose uptake.
rbp <dig> has been recently implicated in systemic insulin sensitivity in rodents and humans, in which elevated serum rbp <dig> levels were associated with reduced expression of glut <dig> in adipocytes, and correlated inversely with peripheral insulin sensitivity.
however, based on current data, the role of rbp <dig> in insulin sensitivity in humans is still controversial and might be restricted to rodent models only.
interspecies differences are known to exist and discrepancies between humans and mice might emphasize the role of non-genetic environmental factors and genetic modifiers in determining the phenotypic variations in rbp <dig> and insulin sensitivity between humans and animal models.
our results in the present study clearly indicate that in hmga1-knockout mice rbp <dig> levels are considerably decreased in serum and in whole liver and adipose tissue extracts, strictly linking hmga <dig> and rbp <dig> expression.
we propose that hmga <dig> deficiency adversely affects rbp <dig> expression and this, in animals with a disadvantageous metabolic risk profile like that observed in the hmga1-knockout mouse model, might reflect an adaptive mechanism to increase glucose uptake and glucose disposal.
consistent with the results obtained in hmga1-deficient mice, rbp <dig> was considerably reduced in cells of both human  and mouse  origin readily expressing rbp <dig>  following perturbation of endogenous hmga <dig> protein expression in cells treated with sirna against hmga <dig>  conversely, an increase in rbp <dig> mrna abundance was observed in both cell lines following forced expression of hmga <dig>  consistently supporting a role for hmga <dig> in the transcriptional activation of the rbp <dig> gene.
these findings were substantiated further by chip analysis, showing that hmga <dig> indeed binds to the rbp <dig> locus in intact living cells.
signal transduction pathways which raise intracellular camp have been reported to have a potential role in the regulation of rbp <dig> gene expression  <cit> .
although the molecular mechanisms underlying this effect remain poorly understood, evidence exists supporting the notion that the regulation of rbp <dig> gene transcription via the camp signaling pathway may be physiologically relevant.
one important physiological condition in which intracellular camp increases is in response to low glucose availability.
in this metabolic setting, a concomitant predominance of circulating counter-regulatory hormones, in particular pancreatic glucagon acting via the camp pathway, induces glycogenolysis and gluconeogenesis in the liver, which produce and release hepatic glucose in the blood.
in this regard, the camp-element-binding protein  has been identified as a critical transcriptional checkpoint which, in response to camp, promotes hepatic glucose output through the synergistic activation of distinct transcriptional effector pathways, which include the ppar gamma coactivator  <dig>  and the nr4a orphan nuclear receptors  <cit> .
in this paper, we report that systemic injection of glucagon to wild-type control mice caused an increase in rbp <dig> mrna and protein expression, along with an increase of both intracellular camp and hmga <dig> levels.
glucagon effects were attenuated in hmga1-deficient mice, supporting a distinct role for hmga <dig> in the regulation of rbp <dig> gene expression and functionally linking this two genes.
as a consequence of the functional link between hmga <dig> and rbp <dig>  a significant increase in glut <dig> mrna and protein was observed in both skeletal muscle and adipose tissues from hmga1-deficient mice compared with controls.
an inverse relationship between rbp <dig> and glut <dig> has been described previously, in the adipose-glut4-/- mouse, in which the decrease in glut <dig> expression that occurs in the fatty tissue of this mutant genotype is accompanied by increased expression and secretion of the fat-derived rbp <dig>  <cit> .
in our model, instead, rbp <dig> expression is genetically impaired due to the lack of hmga <dig> and glut <dig> is increased in both muscle and fat, suggesting that abnormalities in rbp <dig> and/or metabolites of the vitamin a metabolism may directly affect whole-body insulin action and peripheral insulin sensitivity.
in support of this possibility, identification of regulatory single nucleotide polymorphisms in the rbp <dig> gene associated with type  <dig> diabetes has been recently reported  <cit> , while correlations of rbp <dig> with insulin resistance have been confirmed in experimental clinical approaches in humans  <cit> .
although conflicting results have been reported, raising doubt about the postulated relationship of rbp <dig> with insulin sensitivity in humans, our results in hmga1-deficient mice confirm that an inverse correlation indeed exists between rbp <dig> and insulin sensitivity in vivo, in this animal model of diabetes, lending support to previous hypotheses that lowering rbp <dig> levels would be helpful in ameliorating insulin resistance, at least in mice.
overall, our findings provide mechanistic insight into the regulation of glucose uptake and disposal in peripheral tissues, and support further the role of hmga <dig> as a molecule that is likely to be an important emerging factor in the transcriptional activity of genes implicated in the maintenance of glucose homeostasis and metabolic control, such as the insulin receptor gene  <cit> , the leptin gene  <cit> , and, as shown here, the rbp <dig> gene.
apart from the intrinsic biological interest in elucidating the mechanisms leading to improvement in insulin sensitivity, a clear understanding of the molecular process involved is of potential importance in the development of new therapeutic strategies for patients with metabolic disorders such as obesity, diabetes, and other insulin resistant states.
CONCLUSIONS
we propose that hmga <dig> can serve as a modulator of both rbp <dig> gene expression and protein function and represents an important novel mediator of glucose homeostasis in vivo.

 insulin resistance is a metabolic condition found relatively frequently among humans with chronic hyperinsulinemia and in experimental animal models with defective insulin signaling. recently, a link has been established between peripheral insulin sensitivity and the retinol metabolism, and insulin resistance in rodents and humans has been linked to abnormalities of the vitamin a signaling pathway. according to these studies, impaired glucose uptake in adipose tissue results in secondary systemic insulin resistance through release of the adipose-derived serum rbp. however, it is unknown whether rbp effects on insulin sensitivity are vitamin a-dependent or vitamin a-independent. rbp is mainly produced by the liver but also by adipocytes. in plasma, retinol-rbp is found in an equimolar complex with transthyretin, which is a thyroid hormone transport protein that is synthesized in and secreted from the liver. this ternary complex prevents retinol-rbp excretion by the kidney. by impairing insulin signaling in muscle, rbp inhibits glucose uptake and interferes with insulin-mediated suppression of glucose production in the liver, causing blood glucose levels to rise. conversely, mice lacking the rbp gene show increased insulin sensitivity, and normalizing increased rbp serum levels improves insulin resistance and glucose intolerance. hmga is a small basic protein that binds to adenine-thymine rich regions of dna and functions mainly as a specific cofactor for gene activation. hmga by itself has no intrinsic transcriptional activity; rather, it can transactivate promoters through mechanisms that facilitate the assembly and stability of a multicomponent enhancer complex, the so-called enhanceosome, that drives gene transcription. as part of an investigation into the molecular basis regulating the human insulin receptor gene, we previously showed that hmga is required for proper insulin receptor gene transcription. more recently, we showed that loss of hmga expression, induced in mice by disrupting the hmga gene, caused a type 2-like diabetic phenotype, in which, however, impaired glucose tolerance and overt diabetes coexisted with a condition of peripheral insulin hypersensitivity. concomitant insulin resistance and insulin hypersensitivity in peripheral tissues may paradoxically coexist as observed in livers of lipodystrophic and ob/ob mice, as well as in cdk knockout mice with defective pancreatic beta cell development and blunted insulin secretion. the hypothesis that the paradoxical insulin hypersensitivity of hmga1-deficient mice could be due to a deficit, in these animals, of rbp is supported by our data. herein, by employing the hmga1-knockout mouse model, we provide compelling evidence for the identification of a novel biochemical pathway involving hmga and rbp whose activation by the camp pathway may play an important role in maintaining glucose metabolism homeostasis in vivo, in both rodents and humans. the importance of hmga in rbp gene transcription was substantiated in hmga1-deficient mice, in which loss of hmga expression considerably decreased rbp mrna abundance and rbp protein production.  rbp gene transcription is induced by hmga and camp we first performed experiments to see whether hmga had a role in activating the mouse rbp gene promoter at the transcriptional level. to test this possibility, hepg human hepatoma cells and mouse hepa hepatoma cells were cotransfected transiently with mouse rbp4-luc reporter plasmid plus increasing amounts of the hmga expression vector. as shown in figure overexpression of hmga considerably increased rbp4-luc activity in both cell types and this effect occurred in a dose-dependent manner. consistent with these results, rbp mrna abundance was increased in cells overexpressing hmga and was reduced in cells pretreated with sirna targeting hmga, indicating that activation of the rbp gene requires hmga these data were substantiated by chromatin immunoprecipitation assay, showing that binding of hmga to the endogenous rbp locus was increased in whole, intact hepg and hepa cells naturally expressing hmga and was decreased in cells exposed to sirna against hmga. based on these results, in addition to previous observations indicating that camp, or agents which elevate intracellular camp, increase rbp transcript levels, we were interested to see whether a functional link could be established between camp, hmga and rbp to this end, we first confirmed and extended the observation made by jessen and satre that rbp is induced by camp in hepa cells. as measured by northern blot analysis of total rna, rbp mrna increased ≈ 5-fold over the basal level in hepa cells treated with mm 8-bromo camp, a standard concentration for c-amp induction experiments. as shown in figure rbp mrna levels increased starting at h, peaking at h and then declining, suggesting a transient transcriptional stimulation. to establish whether hmga was required for basal and camp-dependent rbp transcription, we transfected the hmga expression vector in hepa cells treated or not with br-camp and rbp protein levels were analyzed h later by western blot. as shown in figure rbp protein expression was enhanced in cells overexpressing hmga and even further in cells treated with camp, in which an increase in hmga protein expression was simultaneously observed, suggesting that induction of rbp by camp may occur, at least in part, through activation of endogenous hmga expression. this hypothesis was supported by the fact that rbp was reduced in camp treated cells in which endogenous levels of hmga were specifically lowered by transfecting cells with hmga antisense expression plasmid. however, further experiments are needed to fully explain the role of camp on hmga expression. the functional significance of hmga in rbp gene expression was confirmed in transient transcription assays in hepa cells, in which overexpression of hmga caused an increase in both basal and camp-induced luc activity from the mouse rbp-luc reporter plasmid. this effect was substantiated in hek- camp-responsive cells, a cell line ideally suited for studying the effects of hmga on transcription since it does not express appreciable levels of this protein. as shown in figure in support of the role that hmga plays in the context of rbp gene, the direct effect of camp was less effective in promoting rbp transcription in hek- cells expressing low levels of hmga becoming considerably higher in cells with forced expression of hmga thus, these data together demonstrate that hmga is of major importance for transcriptional regulation of the rbp gene, and indicate that a functional link exists between camp, hmga and rbp hmga1-deficient mice have reduced expression of rbp in liver and fat tissue and reduced serum rbp levels in the light of the above experimental results, indicating that hmga plays a positive role in rbp gene transcription in living cultured cells, it was interesting to analyze the functional consequences of genetic ablation of hmga on rbp in vivo, in hmga1-knockout mice. to this end, we performed studies aimed at investigating the expression of rbp mrna and protein in hmga1-deficient mice and wild-type controls. as shown in figure rbp mrna was severely attenuated in both liver and fat from hmga1-null mice, and reduced by 50% in hmga heterozygous mutants, as assessed by real-time quantitative polymerase chain reaction. reduced rbp mrna levels in liver and adipose tissue paralleled the decrease in rbp serum levels as detected by western blot analysis of serum samples from age- and body weight-matched mice with diverse genotypes, thereby showing the requirement of hmga for full rbp expression in whole animals. hmga and rbp expression increase in liver and fat of normal mice after intraperitoneal glucagon injection based on our observations in intact cultured cells, indicating a role for the camp signaling pathway in hmga and rbp gene expression, camp-inducible transcriptional activation of the hmga and rbp genes was investigated in vivo, in whole animals, by systemic administration of the intracellular camp-elevating hormone glucagon. under these conditions, glucagon-stimulated camp responses in terms of both hmga and rbp mrna expression were first analyzed in wild-type control mice. consistent with our data in hepa cells, hmga and rbp mrna levels significantly increased in liver and fat of normal mice after intraperitoneal injection of glucagon. time course analyses revealed that the induction and accumulation of hmga mrna preceded the expression of rbp mrna in both tissues. in liver, rbp mrna appeared after that for hmga peaked after h following glucagon injection, and then remained at a plateau. in fat, rbp mrna appeared at h after hmga mrna, peaked after h of glucagon stimulation, and decreased smoothly thereafter. increased levels of hmga and rbp mrnas were paralleled by the increase of hmga and rbp protein expression, as measured by western blot analysis of proteins from liver and fat of glucagon-injected animals. interestingly, when similar experiments were carried out in glucagon injected hmga1-deficient mice, tissue expression of rbp mrna was severely attenuated in liver and fat from heterozygous and hmga1-null animals, thereby indicating that hmga is indeed required for maximal induction of the rbp gene in vivo, in the whole organism, and that the glucagon/adenylate cyclase system regulates both hmga and rbp gene and protein expression. consistent with this conclusion, liver rbp mrna and protein expression levels were lower in fed wild-type mice, becoming higher during fasting, when circulating glucagon increases. as a measure of the glucagon efficacy in glucagon-injected mice, a liver biopsy was taken before and after glucagon injection, and camp levels in liver were determined for both control and hmga1-deficient mice. no substantial difference was found in basal levels of camp. after glucagon injection, hepatic levels of camp increased to μmol/g tissue in control mice, compared with and in hmga1-/- and hmga1+/- mice, respectively. results similar to those shown in the inset of figure were also obtained in epididymal and subcutaneous fat pads from control and mutant animals, thus indicating that the glucagon-stimulated camp synthesis did not differ among mice with diverse genotypes. hmga1-deficient mice have increased glut expression and insulin signaling activity in skeletal muscle and fat systemic insulin resistance has been associated with elevation of serum rbp whereas genetic and pharmacological interventions aimed at decreasing serum rbp levels enhance insulin action and improve insulin sensitivity. increased peripheral insulin sensitivity during insulin-tolerance test was previously observed by us in hmga1-knockout mice. to verify whether a functional link indeed existed between hmga and rbp and whether insulin hypersensitivity in hmga1-deficient mice could be mediated by the hmga1-rbp system, we carried out quantitative measurements of glut mrna transcript abundance. examination by qrt-pcr showed a significant increase of glut transcripts in both skeletal muscle and adipose tissues from hmga1-deficient mice compared with controls. accordingly, immunoblotting of muscle and fat tissue showed a 2- to 3-fold increase of glut in the insulin hypersensitive hmga1-knockout mice compared with controls, clearly indicating that an inverse correlation between rbp and glut indeed exists in vivo, in this animal model of diabetes, in which reduced rbp may contribute to the maintenance of glucose homeostasis by increasing insulin signaling and peripheral insulin sensitivity. in agreement with this interpretation, the activation state of the protein kinase akt, an important downstream target of pi 3-kinase regulating insulin serum effects on glut translocation and carbohydrate metabolism, was increased in mutant animals. as shown in figure basal phospho-akt immunoreactivity was higher in skeletal muscle and adipose tissues from hmga1-deficient mice compared with wild-type controls, and this increase paralleled closely the increase of glut protein in adipose and muscle plasma membranes from heterozygous and homozygous hmga mutants. in line with previous observations on transcriptional repression of the mouse glut gene by camp, endocrine upregulation of glut in hmga1-deficient mice was substantiated further by in vitro experiments, indicating that in isolated adipocytes treated with br-camp, glut mrna was decreased in all three genotypes. a positive correlation of rbp levels with markers of lipid metabolism adversely affecting insulin sensitivity has been reported recently in both clinical and experimental studies. hmga1-knockout mice had lower levels of serum free fatty acids, which might contribute to their improved insulin sensitivity. recombinant rbp injection reduces glut and insulin signaling activity in muscle and fat tissue of hmga1-deficient mice, and attenuates insulin hypersensitivity of these animals to demonstrate that increased insulin sensitivity in mutant mice was directly due to hmga regulation of rbp we determined the effect of recombinant rbp administration on akt phosphorylation and glut protein expression in skeletal muscle from hmga1-deficient mice. as shown in figure akt phosphorylation was reduced in muscle from rbp4-injected mutant animals compared with saline-injected hmga mutants. the reduction in akt phosphorylation in these genotypes correlated inversely with rbp serum levels in the same animals and paralleled the reduction of glut in skeletal muscle and adipose plasma membranes, indicating that, in these conditions, activation of the akt-glut pathway is regulated, at least in part, by circulating rbp plasma insulin levels were slightly higher in rbp4-injected mice, but no significant difference was found. consistent with the condition of insulin hypersensitivity, we previously reported that the glucose-lowering effect of exogenous insulin was enhanced in hmga1-deficient mice during insulin-tolerance test. to support further the role of rbp in insulin hypersensitivity in hmga mutants, we have determined the effect of rbp administration on the glucose fall induced by insulin in these genotypes during itt. as shown in figure injection of human rbp in heterozygous and homozygous hmga mutants caused a less dramatic fall in blood glucose levels, lessening the hypoglycemic response to intraperitoneal insulin observed in the saline-injected animals. thus, taken together, our findings consistently support the role of hmga as a key element in the transcriptional regulation of genes involved in glucose metabolism and add new insights into the compensatory mechanisms that may contribute to counteract insulin resistance in vivo. by directly regulating rbp gene transcription, hmga enhances peripheral insulin sensitivity, ensuring glucose uptake in skeletal muscle. this, if on one hand might represent an adaptive mechanism to ameliorate insulin resistance in animals with a disadvantageous metabolic risk profile, on the other might indicate that the camp/hmga1-mediated rbp expression during fasting may act physiologically to reduce insulin sensitivity in peripheral tissues, thereby contributing to the maintenance of euglycemia under this condition. this was supported by the observation that after an overnight fasting period plasma glucose concentration in wild-type mice was higher than that of hmga1-deficient mice. discussion we have previously shown that loss of hmga protein expression, induced in mice by disrupting the hmga gene, severely decreased insulin receptor expression and phosphorylation in the major targets of insulin action, largely impaired insulin signaling, and reduced insulin secretion, producing a type 2-like diabetic phenotype in which defects in both peripheral insulin sensitivity and pancreatic beta-cell insulin secretion were coexpressed simultaneously. however, despite the severe decrease in insulin receptor signaling and insulin receptor production, the glucose-lowering effect of exogenous insulin was enhanced in hmga1-deficient mice during itt, and the glucose infusion rate necessary to maintain euglycemia was higher in mutant mice during hyperinsulinemic-euglycemic clamp, supporting the existence of alternative pathways of insulin signaling promoting glucose uptake and disposal in certain adverse metabolic conditions such as those found in the hmga1-knockout mouse. the existence of signaling pathways promoting glucose uptake and utilization in peripheral tissues through mechanisms that are independent of insulin has been postulated before, on the basis of experimental observations supporting the existence of molecular circuits/pathways that can compensate for the decrease in insulin-stimulated glucose uptake in vivo, in both animal models and human patients with type diabetes. however, how these compensatory mechanisms are activated has remained hitherto largely undefined. as previously shown, consistent with the ubiquitous distribution of hmga insulin receptor expression was also reduced in pancreatic tissue from hmga1-deficient mice. loss of insulin secretion in response to glucose has been reported in irβ knockout mice with tissue-specific knockout of the insulin receptor in pancreatic beta cells. as in the irβ knockout mice, plasma insulin after glucose challenge was considerably reduced in hmga1-mutant animals, in which the acute first-phase insulin secretory response was severely blunted, indicating a glucose-induced insulin secretory defect. in addition, substantial abnormalities in pancreatic islet morphology and size have been described in hmga1-knockout mice, indicating that decreased insulin secretion in this genotype may also depend on reduced beta-cell mass. thus, defects in both pancreatic beta-cell insulin secretion and peripheral insulin action coexist simultaneously in this knockout mouse model of diabetes, in which activation of compensatory mechanisms to efficiently overcome these metabolic abnormalities may be of vital importance. downregulation of glut in adipose tissue is a typical feature of insulin-resistant states, such as obesity and type diabetes. it has been found that the decrease in glut expression that occurs in the fatty tissue of obese animals and humans is accompanied by increased expression and secretion of the adipocyte-derived rbp fraction, suggesting that rbp production is tightly regulated by adipose tissue glucose uptake. rbp has been recently implicated in systemic insulin sensitivity in rodents and humans, in which elevated serum rbp levels were associated with reduced expression of glut in adipocytes, and correlated inversely with peripheral insulin sensitivity. however, based on current data, the role of rbp in insulin sensitivity in humans is still controversial and might be restricted to rodent models only. interspecies differences are known to exist and discrepancies between humans and mice might emphasize the role of non-genetic environmental factors and genetic modifiers in determining the phenotypic variations in rbp and insulin sensitivity between humans and animal models. our results in the present study clearly indicate that in hmga1-knockout mice rbp levels are considerably decreased in serum and in whole liver and adipose tissue extracts, strictly linking hmga and rbp expression. we propose that hmga deficiency adversely affects rbp expression and this, in animals with a disadvantageous metabolic risk profile like that observed in the hmga1-knockout mouse model, might reflect an adaptive mechanism to increase glucose uptake and glucose disposal. consistent with the results obtained in hmga1-deficient mice, rbp was considerably reduced in cells of both human and mouse origin readily expressing rbp following perturbation of endogenous hmga protein expression in cells treated with sirna against hmga conversely, an increase in rbp mrna abundance was observed in both cell lines following forced expression of hmga consistently supporting a role for hmga in the transcriptional activation of the rbp gene. these findings were substantiated further by chip analysis, showing that hmga indeed binds to the rbp locus in intact living cells. signal transduction pathways which raise intracellular camp have been reported to have a potential role in the regulation of rbp gene expression. although the molecular mechanisms underlying this effect remain poorly understood, evidence exists supporting the notion that the regulation of rbp gene transcription via the camp signaling pathway may be physiologically relevant. one important physiological condition in which intracellular camp increases is in response to low glucose availability. in this metabolic setting, a concomitant predominance of circulating counter-regulatory hormones, in particular pancreatic glucagon acting via the camp pathway, induces glycogenolysis and gluconeogenesis in the liver, which produce and release hepatic glucose in the blood. in this regard, the camp-element-binding protein has been identified as a critical transcriptional checkpoint which, in response to camp, promotes hepatic glucose output through the synergistic activation of distinct transcriptional effector pathways, which include the ppar gamma coactivator and the nr4a orphan nuclear receptors. in this paper, we report that systemic injection of glucagon to wild-type control mice caused an increase in rbp mrna and protein expression, along with an increase of both intracellular camp and hmga levels. glucagon effects were attenuated in hmga1-deficient mice, supporting a distinct role for hmga in the regulation of rbp gene expression and functionally linking this two genes. as a consequence of the functional link between hmga and rbp a significant increase in glut mrna and protein was observed in both skeletal muscle and adipose tissues from hmga1-deficient mice compared with controls. an inverse relationship between rbp and glut has been described previously, in the adipose-glut4-/- mouse, in which the decrease in glut expression that occurs in the fatty tissue of this mutant genotype is accompanied by increased expression and secretion of the fat-derived rbp. in our model, instead, rbp expression is genetically impaired due to the lack of hmga and glut is increased in both muscle and fat, suggesting that abnormalities in rbp and/or metabolites of the vitamin a metabolism may directly affect whole-body insulin action and peripheral insulin sensitivity. in support of this possibility, identification of regulatory single nucleotide polymorphisms in the rbp gene associated with type diabetes has been recently reported, while correlations of rbp with insulin resistance have been confirmed in experimental clinical approaches in humans. although conflicting results have been reported, raising doubt about the postulated relationship of rbp with insulin sensitivity in humans, our results in hmga1-deficient mice confirm that an inverse correlation indeed exists between rbp and insulin sensitivity in vivo, in this animal model of diabetes, lending support to previous hypotheses that lowering rbp levels would be helpful in ameliorating insulin resistance, at least in mice. overall, our findings provide mechanistic insight into the regulation of glucose uptake and disposal in peripheral tissues, and support further the role of hmga as a molecule that is likely to be an important emerging factor in the transcriptional activity of genes implicated in the maintenance of glucose homeostasis and metabolic control, such as the insulin receptor gene, the leptin gene, and, as shown here, the rbp gene. apart from the intrinsic biological interest in elucidating the mechanisms leading to improvement in insulin sensitivity, a clear understanding of the molecular process involved is of potential importance in the development of new therapeutic strategies for patients with metabolic disorders such as obesity, diabetes, and other insulin resistant states.  we propose that hmga can serve as a modulator of both rbp gene expression and protein function and represents an important novel mediator of glucose homeostasis in vivo. 
in comparative studies of normal and mutant mice, glucagon administration caused a considerable upregulation of hmga and rbp expression both at the mrna and protein level in wild-type animals. conversely, in hmga1-knockout mice, basal and glucagon-mediated expression of rbp was severely attenuated and correlated inversely with increased glut mrna and protein abundance in skeletal muscle and fat, in which the activation state of the protein kinase akt, an important downstream mediator of the metabolic effects of insulin on glut translocation and carbohydrate metabolism, was simultaneously increased. we explored the role of these mechanisms in the regulation of glucose homeostasis by studying the hmga1-knockout mouse model. then, by employing the hmga1-knockout mouse model, we provide evidence for the identification of a novel biochemical pathway involving hmga and the rbp whose activation by the camp-signaling pathway may play an essential role for maintaining glucose metabolism homeostasis in vivo, in certain adverse metabolic conditions in which insulin action is precluded. we previously showed that mice lacking the high mobility group a gene developed a type 2-like diabetic phenotype, in which cell-surface insulin receptors were dramatically reduced in the major targets of insulin action, and glucose intolerance was associated with increased peripheral insulin sensitivity. also, the hypothesis that increased insulin sensitivity in hmga1-deficient mice could be related to the deficit of an insulin resistance factor is discussed. this particular phenotype supports the existence of compensatory mechanisms of insulin resistance that promote glucose uptake and disposal in peripheral tissues by either insulin-dependent or insulin-independent mechanisms. we first show that hmga is needed for basal and camp-induced retinol-binding protein gene and protein expression in living cells of both human and mouse origin. further, they provide evidence for the identification of a novel biochemical pathway involving the camp-hmga1-rbp system, whose activation may play a role in glucose homeostasis in both rodents and humans. these results indicate that hmga is an important modulator of rbp gene expression in vivo.
 we previously showed that mice lacking the high mobility group a gene developed a type 2-like diabetic phenotype, in which cell-surface insulin receptors were dramatically reduced in the major targets of insulin action, and glucose intolerance was associated with increased peripheral insulin sensitivity. this particular phenotype supports the existence of compensatory mechanisms of insulin resistance that promote glucose uptake and disposal in peripheral tissues by either insulin-dependent or insulin-independent mechanisms. we explored the role of these mechanisms in the regulation of glucose homeostasis by studying the hmga1-knockout mouse model. also, the hypothesis that increased insulin sensitivity in hmga1-deficient mice could be related to the deficit of an insulin resistance factor is discussed.  we first show that hmga is needed for basal and camp-induced retinol-binding protein gene and protein expression in living cells of both human and mouse origin. then, by employing the hmga1-knockout mouse model, we provide evidence for the identification of a novel biochemical pathway involving hmga and the rbp whose activation by the camp-signaling pathway may play an essential role for maintaining glucose metabolism homeostasis in vivo, in certain adverse metabolic conditions in which insulin action is precluded. in comparative studies of normal and mutant mice, glucagon administration caused a considerable upregulation of hmga and rbp expression both at the mrna and protein level in wild-type animals. conversely, in hmga1-knockout mice, basal and glucagon-mediated expression of rbp was severely attenuated and correlated inversely with increased glut mrna and protein abundance in skeletal muscle and fat, in which the activation state of the protein kinase akt, an important downstream mediator of the metabolic effects of insulin on glut translocation and carbohydrate metabolism, was simultaneously increased.  these results indicate that hmga is an important modulator of rbp gene expression in vivo. further, they provide evidence for the identification of a novel biochemical pathway involving the camp-hmga1-rbp system, whose activation may play a role in glucose homeostasis in both rodents and humans. elucidating these mechanisms has importance for both fundamental biology and therapeutic implications. 