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Recent studies have revealed the impact of antibiotic-induced microbiome depletion (AIMD) on host glucose homeostasis. The kidney has a critical role in systemic glucose homeostasis; however, information regarding the association between AIMD and renal glucose metabolism remains limited. Hence, we aimed to determine the effects of AIMD on renal glucose metabolism by inducing gut microbiome depletion using an antibiotic cocktail (ABX) composed of ampicillin, vancomycin, and levofloxacin in mice. The results showed that bacterial 16s rRNA expression, luminal concentrations of short-chain fatty acids and bile acids, and plasma glucose levels were significantly lower in ABX-treated mice than in vehicle-treated mice. In addition, ABX treatment significantly reduced renal glucose and pyruvate levels. mRNA expression levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase in the renal cortex were significantly higher in ABX-treated mice than in vehicle-treated mice. We further examined the impact of AIMD on the altered metabolic status in mice after ischemia-induced kidney injury. After exposure to ischemia for 60 min, renal pyruvate concentrations were significantly lower in ABX-treated mice than in vehicle-treated mice. ABX treatment caused a more severe tubular injury after ischemia-reperfusion. Our findings confirm that AIMD is associated with decreased pyruvate levels in the kidney, which may have been caused by the activation of renal gluconeogenesis. Thus, we hypothesized that AIMD would increase the vulnerability of the kidney to ischemia-reperfusion injury. NEW & NOTEWORTHY This study aimed to determine the impact of antibiotic-induced microbiome depletion (AIMD) on renal glucose metabolism in mice. This is the first report confirming that AIMD is associated with decreased levels of pyruvate, a key intermediate in glucose metabolism, which may have been caused by activation of renal gluconeogenesis. We hypothesized that AIMD can increase the susceptibility of the kidney to ischemia-reperfusion injury.

There are substantial sex differences in renal structure and ammonia metabolism that correlate with differences in expression of proteins involved in ammonia generation and transport. This study determined the role of testis-derived testosterone in these differences. We studied 4-mo-old male C57BL/6 mice 4 and 8 wk after either bilateral orchiectomy (ORCH) or sham-operated control surgery and determined the effect of testosterone replacement to reverse the effects of ORCH. Finally, we determined the cellular expression of androgen receptor (AR), testosterone's canonical target receptor. ORCH decreased kidney and proximal tubule size, and testosterone replacement reversed this effect. ORCH increased ammonia excretion in a testosterone-dependent fashion; this occurred despite similar food intake, which is the primary component of endogenous acid production. ORCH increased expression of both phospho enol pyruvate, a major ammonia-generating protein, and Na + -K + -2Cl - cotransporter, which mediates thick ascending limb ammonia reabsorption; these changes were reversed with testosterone replacement. Orchiectomy also decreased expression of Na + /H + exchanger isoform 3, which mediates proximal tubule ammonia secretion, in a testosterone-dependent pattern. Finally, ARs are expressed throughout the proximal tubule in both the male and female kidney. Testosterone, possibly acting through ARs, has dramatic effects on kidney and proximal tubule size and decreases ammonia excretion through its effects on several key proteins involved in ammonia metabolism.

Impaired insulin-induced suppression of renal gluconeogenesis could be a risk for hyperglycemia. Diabetes is associated with elevated renal gluconeogenesis; however, its regulation in early insulin resistance is unclear in humans. A noninvasive marker of renal gluconeogenesis would be helpful. Here, we show that human urine exosomes (uE) contain three gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Their protein levels were positively associated with whole body insulin sensitivity. PEPCK protein in uE exhibited a meal-induced suppression. However, subjects with lower insulin sensitivity had blunted meal-induced suppression. Also, uE from subjects with prediabetes and diabetic rats had higher PEPCK relative to nondiabetic controls. Moreover, uE-PEPCK was higher in drug-naïve subjects with diabetes relative to drug-treated subjects with diabetes. To determine whether increased renal gluconeogenesis is associated with hyperglycemia or PEPCK expression in uE, acidosis was induced in rats by 0.28 M NH 4 Cl with 0.5% sucrose in drinking water. Control rats were maintained on 0.5% sucrose. At the seventh day posttreatment, gluconeogenic enzyme activity in the kidneys, but not in the liver, was higher in acidotic rats. These rats had elevated PEPCK in their uE and a significant rise in blood glucose relative to controls. The induction of gluconeogenesis in human proximal tubule cells increased PEPCK expression in both human proximal tubules and human proximal tubule-secreted exosomes in the media. Overall, gluconeogenic enzymes are detectable in human uE. Elevated PEPCK and its blunted meal-induced suppression in human urine exosomes are associated with diabetes and early insulin resistance.

Renal ammonia metabolism has a major role in the maintenance of acid-base homeostasis. Sex differences are well recognized as an important biological variable in many aspects of renal function, including fluid and electrolyte metabolism. However, sex differences in renal ammonia metabolism have not been previously reported. Therefore, the purpose of the current study was to investigate sex differences in renal ammonia metabolism. We studied 4-mo-old wild-type C57BL/6 mice fed a normal diet. Despite similar levels of food intake, and, thus, protein intake, which is the primary determinant of endogenous acid production, female mice excreted greater amounts of ammonia, but not titratable acids, than did male mice. This difference in ammonia metabolism was associated with fundamental structural differences between the female and male kidney. In the female mouse kidney, proximal tubules account for a lower percentage of the renal cortical parenchyma compared with the male kidney, whereas collecting ducts account for a greater percentage of the renal parenchyma than in male kidneys. To further investigate the mechanism(s) behind the greater ammonia excretion in female mice, we examined differences in the expression of proteins involved in renal ammonia metabolism and transport. Greater basal ammonia excretion in females was associated with greater expression of PEPCK, glutamine synthetase, NKCC2, Rhbg, and Rhcg than was observed in male mice. We conclude that there are sex differences in basal ammonia metabolism that involve both renal structural differences and differences in expression of proteins involved in ammonia metabolism.

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.

Circadian rhythms occur in all levels of organization from expression of genes to complex physiological processes. Although much is known about the mechanism of the central clock in the suprachiasmatic nucleus, the regulation of clocks present in peripheral tissues as well as the genes regulated by those clocks is still unclear. In this study, the circadian regulation of gene expression was examined in rat adipose tissue. A rich time series involving 54 animals euthanized at 18 time points within the 24-h cycle (12:12 h light-dark) was performed. mRNA expression was examined with Affymetrix gene array chips and quantitative real-time PCR, along with selected physiological measurements. Transcription factors involved in the regulation of central rhythms were examined, and 13 showed circadian oscillations. Mining of microarray data identified 190 probe sets that showed robust circadian oscillations. Circadian regulated probe sets were further parsed into seven distinct temporal clusters, with >70% of the genes showing maximum expression during the active/dark period. These genes were grouped into eight functional categories, which were examined within the context of their temporal expression. Circadian oscillations were also observed in plasma leptin, corticosterone, insulin, glucose, triglycerides, free fatty acids, and LDL cholesterol. Circadian oscillation in these physiological measurements along with the functional categorization of these genes suggests an important role for circadian rhythms in controlling various functions in white adipose tissue including adipogenesis, energy metabolism, and immune regulation.

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phosphoenolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism.

Sodium arsenite has been demonstrated to alter the expression of genes associated with glucose homeostasis in tissues involved in the pathogenesis of type 2 diabetes; however, the underlying molecular mechanism has not been fully elucidated yet. In this study, we report that the sodium arsenite-induced gene expression of the small heterodimer partner (SHP; NR0B2), an atypical orphan nuclear receptor, regulates the expression of hepatic gluconeogenic genes. Sodium arsenite augments hepatic SHP mRNA levels in an AMP-activated protein kinase (AMPK)-dependent manner. Sodium arsenite activated AMPK and was shown to perturb cellular ATP levels. The arsenite-induced SHP mRNA level was blocked by adenoviral overexpression of dominant negative AMPK (Ad-dnAMPKalpha) or by the AMPK inhibitor compound C in hepatic cell lines. We demonstrated the dose-dependent induction of SHP mRNA levels by sodium arsenite and repressed the forskolin/dexamethasone-induced gene expression of the key hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Ad-dnAMPKalpha blocked the repressive effects of arsenite-induced SHP on PEPCK and G6Pase. Sodium arsenite inhibited the promoter activity of PEPCK and G6Pase, and this repression was abolished by small interfering (si)RNA SHP treatments. The knockdown of SHP expression by oligonucleotide siRNA SHP or adenoviral siRNA SHP released the sodium arsenite-mediated repression of forskolin/dexamethasone-stimulated PEPCK and G6Pase gene expression in a variety of hepatic cell lines. Results from our study suggest that sodium arsenite induces SHP via AMPK to inhibit the expression of hepatic gluconeogenic genes and also provide us with a novel molecular mechanism of arsenite-mediated regulation of hepatic glucose homeostasis.

Individuals born with a low birth weight (LBW) have an increased prevalence of type 2 diabetes, but the mechanisms responsible for this association are unknown. Given the important role of insulin resistance in the pathogenesis of type 2 diabetes, we examined insulin sensitivity in a rat model of LBW due to intrauterine fetal stress. During the last 7 days of gestation, rat dams were treated with dexamethasone and insulin sensitivity was assessed in the LBW offspring by a hyperinsulinemic euglycemic clamp. The LBW group had liver-specific insulin resistance associated with increased levels of PEPCK expression. These changes were associated with pituitary hyperplasia of the ACTH-secreting cells, increased morning plasma ACTH concentrations, elevated corticosterone secretion during restraint stress, and an approximately 70% increase in 24-h urine corticosterone excretion. These data support the hypothesis that prenatal stress can result in chronic hyperactivity of the hypothalamic-pituitary-adrenal axis, resulting in increased plasma corticosterone concentrations, upregulation of hepatic gluconeogenesis, and hepatic insulin resistance.

Isoleucine, a branched chain amino acid, plays an important role in the improvement of glucose metabolism as evidenced by the increase of insulin-independent glucose uptake in vitro. This study evaluated the effect of isoleucine on glucose uptake and oxidation in fasted rats and on gluconeogenesis in vivo and in vitro. Oral administration of isoleucine decreased the plasma glucose level by 20% and significantly increased muscle glucose uptake by 71% without significant elevation of the plasma insulin level compared with controls at 60 min after administration. Furthermore, expiratory excretion of 14CO2 from [U-14C]glucose in isoleucine-administered rats was increased by 19% compared with controls. Meanwhile, isoleucine decreased AMP levels in the liver but did not affect hepatic glycogen synthesis. Under insulin-free conditions, isoleucine significantly inhibited glucose production when alanine was used as a glucogenic substrate in isolated hepatocytes. This inhibition by isoleucine was also associated with a decline in mRNA levels for phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) and a decreased activity of G6Pase in isolated hepatocytes. These findings suggest that a reduction of gluconeogenesis in liver, along with an increase of glucose uptake in the muscle, is also involved in the hypoglycemic effect of isoleucine. In conclusion, isoleucine administration stimulates both glucose uptake in the muscle and whole body glucose oxidation, in addition to depressing gluconeogenesis in the liver, thereby leading to the hypoglycemic effect in rats.

Blood pH controls the activity of important regulatory enzymes in the metabolism. Serine dehydratase (SerDH) transforms l-serine into pyruvate and ammonium and is involved in the regulation of gluconeogenesis from serine in the rat liver. In this work, we investigate the effect of chronic metabolic acidosis on the kinetics, specific protein level, tissue location, and mRNA levels of rat liver SerDH. Experimental acidosis was induced in rats by ingestion of 0.28 M ammonium chloride solution for 10 days. Acidosis significantly (P<0.05) decreased SerDH activity at all substrate concentrations assayed. Moreover, the Vmax value was 38.50+/-3.51 mU/mg (n=7) of mitochondrial protein in the acidotic rats and 92.49+/-6.79 mU/mg (n=7) in the control rats. Western blot analysis revealed a significant reduction (14%) in the level of SerDH protein content in the rat liver during acidosis. Immunohistochemical analysis showed that SerDH location did not change in response to chronic metabolic acidosis and confirmed previous results on SerDH protein levels. Moreover, the SerDH mRNA level, estimated by RT-PCR, was also significantly 33.8% lower than in control. These results suggest that during experimental acidosis a specific repression of rat-liver SerDH gene transcription could result, lowering the amount and activity of this enzyme. The changes found in SerDH expression are part of an overall metabolic response of liver to maintain acid-base homeostasis during acidosis.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a rate-limiting step in hepatic and renal gluconeogenesis. In the kidney, PEPCK expression is enhanced during metabolic acidosis and in response to ANG II and parathyroid hormone. The effect of the latter hormone is mediated, in part, by cAMP. Treatment of subconfluent cultures of LLC-PK1-F+ cells, a gluconeogenic line of porcine proximal tubule-like cells, with cAMP produces a pronounced increase in the level of PEPCK mRNA. The luciferase activity of pLuc/3'-PCK-1, a reporter construct that contains the 3'-UTR of the PEPCK mRNA, was increased three- to fourfold by coexpression of the catalytic subunit of protein kinase A (PKA). This result indicates that cAMP-dependent stabilization may contribute to the increased expression of PEPCK mRNA in LLC-PK1-F+ cells. Various pLuc/3' constructs containing different segments of the 3'-UTR of PEPCK mRNA were used to map the cAMP response to two segments that were previously shown to bind AUF1 and to function as instability elements. A tetracycline-responsive promoter system was used to quantify the effect of forskolin on the half-lives of chimeric beta-globin-PEPCK (TbetaG-PCK) mRNAs. The half-life of the labile betaG-PCK-1 mRNA was increased eightfold by addition of forskolin. In contrast, the half-lives of the constructs containing the individual instability elements were increased only twofold. Therefore, the multiple instability elements present within the 3'-UTR may function synergistically to mediate both the rapid degradation and the cAMP-induced stabilization of PEPCK mRNA. The latter process may result from a PKA-dependent phosphorylation of AUF1.

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.

Alcohol exposure during pregnancy is associated with fetal growth restriction and programs the offspring to insulin resistance later in life. The underlying mechanisms are still uncertain, but a dysregulation of gluconeogenesis and adipose hormones may be contributory. Newborn rats from dams that had been given ethanol (EtOH) or water (controls) during pregnancy were studied. Adiponectin mRNA was determined in subcutaneous fat by RT-PCR, and serum adiponectin was measured by RIA. Subsets of rats were killed before and after intraperitoneal administration of insulin, to determine, by RT-PCR, the hepatic expression of gluconeogenic enzymes and that of the transcription factor peroxisome proliferator-activated receptor-coactivator (PGC)-1, which promotes gluconeogenesis. EtOH offspring had delayed hypoglycemic response to insulin but normal adiponectin mRNA and serum levels compared with controls. The inhibitory response of the gluconeogenic enzyme phosphoenol- pyruvate carboxykinase (PEPCK) and PGC-1 mRNAs to insulin was blunted in EtOH offspring compared with controls. The data suggest that intrauterine EtOH exposure causes insulin resistance of genes for PGC-1 and PEPCK early in life.

LLC-PK(1)-FBPase(+) cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK(1)-FBPase(+) cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK(1)-FBPase(+) cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha-ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK(1)-FBPase(+) cells.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK(-2300)Luc reporter constructs in LLC-PK(1)-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK(1)-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta. Treatment of cells with cAMP did not affect the expression of C/EBPbeta, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK(-2300)Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta, but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.