DGKζ deficiency protects against peripheral insulin resistance and improves energy metabolism

Diacylglycerol kinases (DGKs) regulate the balance between diacylglycerol (DAG) and phosphatidic acid. DGKζ is highly abundant in skeletal muscle and induces fiber hypertrophy. We hypothesized that DGKζ influences functional and metabolic adaptations in skeletal muscle and whole-body fuel utilization. DAG content was increased in skeletal muscle and adipose tissue, but unaltered in liver of DGKζ KO mice. Linear growth, body weight, fat mass, and lean mass were reduced in DGKζ KO versus wild-type mice. Conversely, male DGKζ KO and wild-type mice displayed a similar robust increase in plantaris weight after functional overload, suggesting that DGKζ is dispensable for muscle hypertrophy. Although glucose tolerance was similar, insulin levels were reduced in high-fat diet (HFD)-fed DGKζ KO versus wild-type mice. Submaximal insulin-stimulated glucose transport and p-Akt Ser473 were increased, suggesting enhanced skeletal muscle insulin sensitivity. Energy homeostasis was altered in DGKζ KO mice, as evidenced by an elevated respiratory exchange ratio, independent of altered physical activity or food intake. In conclusion, DGKζ deficiency increases tissue DAG content and leads to modest growth retardation, reduced adiposity, and protection against insulin resistance. DGKζ plays a role in the control of growth and metabolic processes, further highlighting specialized functions of DGK isoforms in type 2 diabetes pathophysiology.

total DGK activity in skeletal muscle from type 2 diabetic patients and animal models of the disease (9). Type II DGK isoenzymes (, , and ) are characterized by a pleckstrin homology domain at the N terminus that may be important for subcellular localization (4). Elevated levels of glucose (9) or monounsaturated free fatty acids (10) reduce DGK abundance and total DGK activity, implicating that systemic factors associated with type 2 diabetes attenuate DGK signaling and localization. Overexpression of DGK in myotubes enhances glucose uptake (11), whereas DGK haploinsufficiency in mice leads to peripheral insulin resistance, metabolic inflexibility, and age-dependent obesity, concomitant with DAG-induced PKC activity and attenuation of insulin signaling (9). The type III isoenzyme, DGK, contains a hydrophobic segment to confer membrane localization (4). DGK influences skeletal muscle levels of unsaturated and saturated DAG species and alters glucose tolerance and whole-body lipid oxidation (12). Thus, abundance, localization, and substrate specificity of each DGK isoform, as well as the balance between DAG and PA species, may influence cellular processes as diverse as insulin secretion, glucose metabolism, and energy homeostasis.
The role of type IV (DGK and DGK) and type V (DGK) DGKs in metabolic disease is uncharted. DGK and DGK have a myristoylated alanine-rich C kinase substrate (MARCKS) homology phosphorylation site domain that functions as a localization signal, as well as four ankyrin repeats and a carboxy terminal PDZ binding domain (4). DGK is abundantly expressed in brain, skeletal muscle, heart, and pancreas (13). Skeletal and cardiac tissuespecific DGK variants are predominantly localized in the nucleus (14,15), consistent with a role for this isoenzyme in myogenic differentiation (15) and cardiac and skeletal muscle hypertrophy (16,17). While DGK has been proposed as a potential therapeutic target to block cardiac dysfunction and prevent congestive heart failure (18), the role of this isoenzyme in insulin resistance and type 2 diabetes is unknown. The aim of this study was to determine the role of DGK in functional and metabolic adaptions in skeletal muscle and whole-body glucose and energy homeostasis. As overexpression of DGK induces skeletal muscle fiber hypertrophy (16), we hypothesized that ablation of DGK may influence functional and metabolic properties of skeletal muscle.

Animals
DGK KO mice were generated on a mixed C57BL/6×129X1/ SvJ background, as described earlier (19) and kindly provided by Dr. Matthew K. Topham (University of Utah, Salt Lake City, UT). We studied DGK KO mice on an inbred strain (C57BL/6) generated by successive backcross breeding. The expression of DGK, -, -, and -, DGK isoforms known to be expressed in skeletal muscle and adipose tissue (20), was unaltered between DGK KO and wild-type littermates (data not shown). Thus, other DGK isoforms do not appear to compensate for the loss of DGK in this model. Age-and sex-matched wild-type littermates were used as controls. Animals were maintained in a temperature controlled facility on a 12/12 light/dark cycle with free access to food and water. Mice were fed normal chow or high-fat diet (HFD; 54.8% energy from fat) from 5 weeks of age for 12 weeks, as described (12). Body weight was measured weekly. For terminal experiments, animals were anesthetized and liver, gonadal fat, and skeletal muscle were collected for biochemical assays, as described below. Several types of skeletal muscle were sampled. In mouse models, extensor digitorum longus (EDL) and gastrocnemius are fast twitch muscles, predominantly composed of type IIB and IIDB fibers, whereas soleus is a slow twitch muscle, predominantly composed of type IIA fibers (21). We used EDL and gastrocnemius interchangeably and soleus, as these muscle types provide a broad representation of the muscles of the whole body in the mouse. For assays requiring large amounts of tissue, we selected gastrocnemius over EDL. The regional animal ethical committee (Stockholm, Sweden) approved all experimental procedures.

Glucose tolerance test
Glucose (2 g/kg body weight) was administered by intraperitoneal injection to 4 h-fasted (15 weeks of age) chow-or HFD-fed mice. Blood was sampled via the tail vein to assess glucose (One Touch Ultra glucose meter; Lifescan, Milpitas, CA) and insulin (Insulin ELISA kit; Crystal Chem Inc., Downers Grove, IL) concentration.

Body composition
Lean mass and fat mass were assessed in conscious mice (14 weeks of age) using the EchoMRI-100 system (Echo Medical Systems, Houston, TX).

Whole-body energy homeostasis
Food intake, oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), respiratory exchange ratio (RER), and locomotor activity were measured using a Comprehensive Lab Animal Monitoring System (Columbus Instruments, Columbus, OH) as described (9).

Ex vivo lipolysis in gonadal adipose tissue
Mice were fasted for 4 h, anesthetized (Avertin, 2,2,2-tribromo ethanol 99% and tertiary amyl alcohol, at 15-17 l/g body weight, ip), and gonadal adipose tissue was collected. Tissue (20 mg) was incubated in the absence or presence of isoprenaline (10 6 M) for 90 min at 37°C in D-PBS supplemented with 2% RIA-grade BSA. The tissue was removed and the glycerol concentration in the medium was determined. Glycerol release into the medium was analyzed as a marker of lipolysis after stimulation with isoprenaline. Glycerol was measured using a Zenbio glycerol analysis kit (Research Triangle Park, NC).

Euglycemic-hyperinsulinemic clamp
Glucose turnover rate was measured in conscious HFD-fed mice (17 weeks of age) under basal and euglycemic-hyperinsulinemic conditions, as described (9). Hepatic glucose production was determined by subtracting the average glucose infusion rate at the steady state from the glucose utilization.

Induction of skeletal muscle hypertrophy
Functional overload was performed by surgical bilateral removal of soleus and gastrocnemius muscles, as described (23,24). Sham-operated mice in which the plantaris, soleus, and gastrocnemius muscles were separated from each other with a forceps were used as a control. Fourteen days after the surgery, fed mice were anesthetized with Avertin (0.02 ml/g; 2.5% solution of 99% 2,2,2-tribromo ethanol and tertiary amyl alcohol) and plantaris muscles were weighed and stored at 80°C until further processing. Plantaris muscle was studied because this particular muscle undergoes marked hypertrophy after surgical removal of the gastrocnemius and soleus muscles from the mouse leg.

DAG content
Total tissue DAG content was determined using a mouse DAG ELISA kit (Cusabio, Wuhan, China).

Statistical analysis
Statistical analysis was performed using Student's t-test, two-way ANOVA, or repeated measures two-way ANOVA. Post hoc analysis with Sidak's testing was performed when a main effect was detected. All data are reported as mean ± SEM. Differences between groups were considered statistically significant at P < 0.05.

DGK alters DAG content in skeletal muscle and adipose tissue, but not liver
Total DAG content was determined in gastrocnemius muscle, gonadal adipose tissue, and liver from male and female DGK KO and wild-type mice fed either chow or HFD ( Fig. 1A-F). DAG content was increased in skeletal muscle (Fig. 1A, D) and gonadal adipose tissue (Fig. 1B, E), but unaltered in liver (Fig. 1C, F), from male and female DGK KO mice irrespective of diet. The tissue-specific changes in DAG content in DGK KO versus wild-type mice correspond with the mRNA profile of DGK (13,14,25), with high levels in skeletal muscle, modest levels in gonadal adipose tissue, and low levels in liver.

DGK influences body weight and growth curves
Body weight of chow-or HFD-fed male and female DGK KO and wild-type mice was determined at 5 weeks of age and measured once a week for 10 weeks (Fig. 1G, K).
Irrespective of diet, body weight of DGK KO mice was reduced compared with age-and sex-matched wild-type mice. Over this 10 week period, DGK KO mice gained less weight on HFD as compared with wild-type mice, irrespective of sex ( Fig. 1H, L). Differences in body weight were more pronounced in female DGK KO mice compared with male DGK KO mice, particularly under chow-fed conditions. Linear growth (snout to anus length) was reduced in male and female DGK KO mice (Fig. 1I, M). Body composition analysis was performed at 14 weeks of age. Fat mass was reduced in both male and female DGK KO mice irrespective of diet (Fig. 1J, N). Lean body mass was reduced in chow-fed female DGK KO mice, but not chow-fed male DGK KO mice (Fig.  1J, N). Thus, DGK deficiency is associated with growth retardation, a moderate reduction in lean mass, and a profound reduction in adiposity, even in HFD-challenged mice.

HSL protein abundance and phosphorylation in white adipose tissue
To gain insight into the mechanism for the reduction in adiposity in DGK KO mice, abundance and phosphorylation of proteins controlling lipolysis were assessed in adipose tissue from 4 h-fasted HFD-fed male and female DGK KO and wild-type mice. HSL is a key enzyme involved in the regulation of lipolysis in white adipose tissue. Phosphorylation of HSL on Ser 660 , a major PKA phosphorylation site, tended to increase in white gonadal adipose tissue from DGK KO mice ( Fig. 2A, B), whereas protein abundance of HSL was unaltered ( Fig. 2C, D). Ex vivo lipolysis was assessed in gonadal adipose tissue (Fig. 2E, F). A trend for increased basal and isoprenaline-stimulated lipolysis was observed in male, but not female, DGK KO mice. These findings indirectly suggest a modest increase in lipolytic activity. Protein abundance of ATGL, a lipid droplet-bound coactivator (CGI58), and enzymes involved in triglyceride synthesis (DGAT1 and DGAT2) were unaltered in gonadal white adipose tissue from DGK KO mice (data not shown). Protein abundance of ACC and FASN, two key enzymes involved in lipogenesis, as well as ACC Ser 79 phosphorylation, was unaltered in gonadal adipose tissue from DGK KO mice (data not shown).

Role of DGK in skeletal muscle growth and hypertrophy
As body weight and lean body mass were reduced in DGK KO mice, we hypothesized that DGK may play a critical role in skeletal muscle growth and hypertrophy. Thus, we measured protein abundance of myogenin in skeletal muscle, given the requirement of this transcription factor in myocyte fusion and myotube formation. Interestingly, myogenin KO mice have reduced body size, implicating a role for this myogenic regulatory factor in regulating body homeostasis (26). We found myogenin abundance was reduced in EDL and soleus muscle from chow-fed female DGK KO mice, but not chow-fed male DGK KO mice (Fig. 3A-D). To investigate the role of DGK in the dynamic control of skeletal muscle mass, male DGK KO and wild-type mice were subjected to functional overload to induce muscle hypertrophy. DGK KO and wild-type mice displayed a robust increase in absolute and relative plantaris weight compared with respective control sham-operated mice (Fig. 3E, F). Plantaris muscle weight was unaltered between DGK KO and wild-type mice after functional overload. Our results suggest that DGK is dispensable for skeletal muscle hypertrophy following a 14-day functional overload. Plantaris muscles of control sham-operated DGK KO mice were slightly lighter as compared with wild-type control mice. This difference was not preserved following functional overload, indicating that plasticity and the hypertrophic response of skeletal muscle is retained in DGK KO mice. . Two-way ANOVA or repeated measures two-way ANOVA with Sidak's post hoc testing. ‡ P < 0.05 overall genotype difference, † P < 0.05 overall diet effect, # P < 0.05 interaction; *P < 0.05 versus wild-type mice on same diet. HFD-fed DGK KO and wild-type mice irrespective of sex (Fig. 4A, B). However, insulin levels measured in the fasting state and 15 min into the glucose tolerance test were reduced in male and female HFD-fed DGK KO versus wild-type mice (Fig. 4C, D), suggesting that insulin sensitivity is enhanced.
To test the hypothesis that DGK deficiency alters insulin sensitivity, we measured insulin-stimulated glucose transport and signal transduction in isolated skeletal muscle from HFD-fed DGK KO and wild-type mice. EDL muscle was incubated ex vivo in the presence or absence of a sub-maximal dose of insulin (0.36 nmol/l) and glucose transport was assessed. Basal glucose transport was similar between HFD-fed DGK KO and wild-type mice, irrespective of sex (Fig. 4E, F). Insulin-stimulated glucose uptake was impaired in EDL muscle from HFD-fed male and female wildtype mice, consistent with our earlier finding that HFD impairs insulin sensitivity of glucose transport in skeletal

Role of DGK in glucose homeostasis
Elevated DAG content in skeletal muscle, adipose tissue, and liver has been implicated in development of insulin resistance (27). Thus, we determined the role of DGK in the control of glucose homeostasis in male and female chow-fed mice. We found that fasting glucose levels and glucose tolerance curves were similar between DGK KO and wild-type mice irrespective of sex (Fig. 4A, B). Insulin levels measured in the fasting state and 15 min into the glucose tolerance test were also similar between DGK KO and wild-type mice irrespective of sex (Fig. 4C, D). Thus, DGK is dispensable for normal glucose homeostasis in chow-fed mice, despite the elevation of total DAG content in skeletal muscle and gonadal adipose tissue. We next determined the role of DGK in the control of glucose tolerance in HFD-fed male and female mice. Similar to our findings in chow-fed mice, glucose tolerance was unaltered between Student's t-test. Gonadal adipose tissue was obtained from 17-week-old 4 h-fasted DGK KO and wild-type mice after 12 weeks on HFD and incubated ex vivo in the absence (basal) or presence of isoprenaline (10 6 M) for measurement of glycerol release into the medium in male (E) and female (F) mice (n = 5-9 mice). Twoway ANOVA with Sidak's post hoc testing. † P < 0.05 overall isoprenaline effect. muscle (28). In contrast, insulin-stimulated glucose transport was enhanced in EDL muscle from HFD-fed male and female DGK KO mice (Fig. 4E, F). Consistent with our results for glucose transport, insulin-stimulated phosphorylation of Akt on Ser 473 (Fig. 4G, H) and Thr 308 (Fig. 4I, J) was enhanced in EDL muscle from HFD-fed male and female DGK KO mice. Collectively, our results suggest that DGK deficiency protects against peripheral insulin resistance in HFD-fed mice, despite elevated DAG content in peripheral tissues.

DGK drives whole body carbohydrate metabolism
We next measured several metabolic parameters in vivo in male and female HFD-fed mice over 3 days, including food consumption, locomotor activity, VO 2 , VCO 2 , and RER. RER was elevated in DGK KO mice compared to wild-type mice during the light and dark phases, but not during a 12 h fasting period. VO 2 was higher in DGK KO mice (data not shown). These data suggest that energy expenditure is upregulated in DGK KO mice, with carbohydrates serving as a preferred energy substrate during rest and active phases, respectively (Fig. 5A, B). Food intake [recorded as absolute amount (Fig. 5C, D) or as a percentage of total body weight (data not shown)] and locomotor activity (Fig. 5E, F) were unaltered between HFD-fed DGK KO and wild-type mice, irrespective of sex. Basal and insulin-mediated peripheral glucose utilization and hepatic glucose production in conscious mice assessed in vivo during a euglycemic-hyperinsulinemic clamp were similar between genotypes (Fig. 5G, H). We also found that tissue weight, as well as mRNA expression of UCP1 and PGC1, was unaltered in brown adipose tissue of wild-type versus DGK KO mice (data not shown). These results suggest that the elevated energy expenditure in DGK KO mice throughout the light and dark phases is independent of changes in physical activity, food intake, and thermogenesis, but may occur from a metabolic switch that promotes increased glucose oxidation. DISCUSSION DGK has been implicated in several pathophysiological conditions, including cancer, cardiovascular disorders, autoimmune diseases, and obesity (29), but the physiological role of this enzyme in functional and metabolic adaptations of skeletal muscle and whole-body glucose and energy homeostasis is unknown. Here, we provide evidence that lifelong deficiency of DGK leads to modest growth retardation, reduced adiposity, and protection against peripheral insulin resistance in mice fed a HFD, despite elevated DAG content in peripheral tissues.
DGK isoforms have unique subtype-specific structural and functional domains, suggesting that each subfamily plays a distinct physiological role. DGK is characterized by the presence of two C1 domains, a MARCKS homology phosphorylation site domain that contains a nuclear localization signal and acts as a substrate for conventional PKCs, a conserved catalytic domain that contains a nuclear export signal, and four ankyrin repeats (30). The presence of nuclear localization and nuclear export signals suggest that DGK plays a role in the regulation of nuclear events and gene expression, either by acting as a signal transducer, interacting as a component of large protein complexes that bring together lipid metabolism enzymes, or by directly altering the local concentration of DAG and PA. Our finding that DGK influences body weight, lean body mass, and adiposity support a role of this isoform in growth, development, and metabolism. DGK affects cell cycle and skeletal muscle differentiation (15,(31)(32)(33). In mouse myoblasts, DGK expression is increased during myogenic differentiation and associated with the nuclear matrix, implicating a role in DNA replication, gene expression, and protein phosphorylation (33). Gene silencing of DGK impairs skeletal muscle differentiation, as evidenced by reduced protein abundance of myogenin (33). Conversely, gene silencing of DGK, a type II subfamily member, did not alter skeletal muscle differentiation (33) or lean body mass (9), indicating DGK isoform- were assessed during the intraperitoneal glucose tolerance tests. EDL muscle was obtained from 17-week-old 4 h-fasted DGK KO and wild-type mice after 12 weeks on HFD and incubated ex vivo in the absence (basal) or presence of insulin (0.36 nmol/l) for measurement of glucose transport (E, F) and Akt Ser 473 and Thr 308 phosphorylation (G-J) in male (E, G, I) and female (F, H, J) mice (n = 9-13 mice). Two-way ANOVA or repeated measures two-way ANOVA with Sidak's post hoc testing. C, D: ‡ P < 0.05 overall genotype difference, † P < 0.05 overall diet effect, # P < 0.05 interaction; *P < 0.05 versus wild-type mice on same diet. E-J: ‡ P < 0.05 overall genotype difference, † P < 0.05 overall insulin effect, # P < 0.05 interaction; *P < 0.05 versus wild-type mice of same condition. specific regulation. We found that myogenin expression was reduced in skeletal muscle from chow-fed female DGK KO mice, but not male DGK KO mice. Myogenin is essential for skeletal muscle development and viability during fetal development, but dispensable when deleted after birth (26). An earlier description of another cohort of these DGK KO mice suggests muscle to body weight ratio, muscle fiber type distribution, and histological appearance are unaltered (16). While these findings are inconsistent with our results, several differences exist between these studies. For example, we studied male and female mice on a C57BL/6 background at 15-17 weeks of age, whereas earlier workers (16) studied 8-to 10-week-old mice on a mixed C57BL/6×129X1/SvJ background of an undetermined sex and results were compared against wild-type FVB/N and C57BL/6 mice, rather than wild-type littermates.
DGK overexpression attenuates cardiac hypertrophy in response to G-protein-coupled receptor agonists or pressure overload (34)(35)(36) and inhibits myocardial atrophy in streptozotocin-induced diabetic mice (17). Skeletal muscle hypertrophy involves an increase in mass and cross-sectional area of individual skeletal muscle fibers. Given the proposed role of DGK in skeletal muscle differentiation and cell growth (32), we subjected DGK KO mice to functional overload in order to determine the requirement of DGK for hypertrophy in mature adult skeletal muscle. Functional overload mimics effects of resistance exercise training and drives a hypertrophic response on muscle growth. Overexpression of DGK in tibialis anterior muscle leads to an activation of PA-mTOR signaling and increases skeletal muscle fiber size (16). While DGK is a component of a mechanosensitive signaling cascade that regulates skeletal muscle mass, we found that DGK was not required for skeletal muscle hypertrophy following a 14 day functional overload. While DGK appears to play a role in myogenic differentiation and growth, skeletal muscle hypertrophy arising from functional overload clearly involves the interplay between multiple signal transducers and pathways to ensure skeletal muscle plasticity (37).
HFDs are commonly used in animal models as an environmental stressor to rapidly increase body weight and reduce whole-body insulin sensitivity. In skeletal muscle, chronic HFD impairs insulin signal transduction and glucose uptake in wild-type mice (28). We found that DGK deficiency protects against peripheral insulin resistance in HFD-fed mice, despite elevated DAG content in peripheral tissues. Insulin sensitivity was increased in DGK KO mice in isolated skeletal muscle and during the glucose tolerance test. Conversely, insulin responsiveness, as determined during the euglycemic-hyperinsulinemic clamp, was similar between DGK KO and wild-type mice, suggesting that the maximal rate of glucose uptake was unaltered. The metabolic phenotype and increased insulin sensitivity of DGK KO mice was unexpected, given our earlier observation that deficiency of DGK, a member of the type II subfamily of DGKs, also increased DAG content; but in this model, peripheral insulin sensitivity, insulin signaling, and glucose transport were reduced and age-dependent obesity was apparent (9). Deficiency of the type III subfamily member, DGK, also influences the balance of DAG species implicated in the development of peripheral insulin resistance in skeletal muscle (12). Although insulin sensitivity was un- . Whole-body glucose utilization in DGK KO and wild-type mice. Basal and insulin-stimulated (clamp) whole-body glucose utilization and hepatic glucose production (HGP) were determined during a euglycemic-hyperinsulinemic clamp in 4 h-fasted mice at age 17 weeks after 12 weeks on HFD in male (G) and female (H) mice (n = 6-7). Two-way ANOVA with Sidak's post hoc test ing. ‡ P < 0.05 overall genotype effect, † P < 0.05 overall diet effect, # P < 0.05 interaction; *P < 0.05 versus wild-type mice of same time point. altered in DGK-deficient mice, whole-body RER was reduced and abundance of mitochondrial markers was increased, despite the elevated DAG levels, indicating a greater reliance on fat oxidation and intracellular lipid metabolism (12). These results with isoform-specific DGK KO mouse models challenge the notion that elevated levels of total DAG content in skeletal muscle and adipose tissue directly impair whole-body glucose and energy homeostasis.
Our finding that DGK KO mice are protected against the development of insulin resistance, despite elevated tissue levels of DAG is a paradox. Several observational studies in type 2 diabetic or obese individuals suggest that total DAG content in skeletal muscle is not consistently elevated despite severe insulin resistance (38)(39)(40). Moreover, skeletal muscle total DAG content is elevated in insulin-sensitive endurance-trained athletes (34,39). Thus chain length, the degree of fatty acid saturation, and the intracellular localization of specific DAG species, rather than elevations in total DAG content, may play an important role in the development of insulin resistance (41)(42)(43). Moreover, multiple isomers of DAG exist, and not all DAG species appear to provoke insulin resistance (42). Thus, we cannot exclude the possibility that DGK KO mice may accumulate 1,3-DAG, an isomer that promotes insulin sensitivity (44), rather than 1,2-DAG, an isomer that activates PKC (45), which could also explain the apparent paradox. Alterations in the intracellular localization of PA may also influence signal transduction and skeletal muscle metabolism (46). DGK isoforms appear to influence growth and metabolism by distinct mechanisms depending on structural diversity and relative tissue-specific abundance, as well as localization and particular lipid species metabolized.
DGK KO mice are protected against the development of insulin resistance on a HFD. These metabolic improvements may be partly related to the reduction in fat mass, possibly due to altered HSL activity and lipolysis in gonadal adipocytes. We also found a shift in RER, suggesting increased energy expenditure in DGK-deficient mice, with carbohydrates serving as the preferred energy substrate during both the light and dark cycle. The apparent greater reliance for carbohydrate as an energetic source in DGK-deficient mice is corroborated by the increased insulin-stimulated glucose transport in skeletal muscle and reduced insulin levels during the glucose tolerance test, suggesting that DGK deficiency enhances insulin sensitivity. The trend for increased p-HSL Ser 660 abundance and lipolysis in adipose tissue, suggesting increased overall lipolysis, concomitant with increased glucose uptake in skeletal muscle, may appear paradoxical; however, increase of fatty oxidation promotes an increase in acetyl-CoA, which is transformed into citrate. A fraction of citrate is transported to the cytosol where it inhibits PFK-1 activity, leading to decreased glucose transport and oxidation (47,48). Our results suggest that DGK deficiency in adipose tissue and skeletal muscle influences glucose and energy homeostasis. Nevertheless DGK is also expressed in distinct regions of the brain, in particular hypothalamic neurons, and via its ankyrin repeats, interacts with the cytoplasmic portion of the leptin receptor (Ob-Rb) (49). Paradoxically, an inverse relationship between hypothalamic DGK mRNA levels and body fat has been reported in HFD-fed rodents, leading to the hypothesis that reductions in DGK activity in the hypothalamus may lead to obesity. The role of hypothalamic DGK is likely to be complex given that we observed that DGK deficiency was associated with leanness and enhanced skeletal muscle insulin sensitivity, rather than the predicted (49) obesity and insulin resistance phenotype. Notably, the augmentation in whole-body energy expenditure occurred without alterations in physical activity or behavioral defects. Our results suggest that a simultaneous increase in both glucose and fatty acid oxidation in peripheral tissues contributes to the enhanced insulin sensitivity in DGK KO mice. Given our results, complementary approaches to examine the tissue-specific role of DGK, particularly in brain, skeletal muscle, heart, and pancreas, may be warranted. However, our study of whole-body DGK KO mice provides important insight to the role of this gene in the regulation and whole-body metabolism and growth.
In conclusion, life-long deficiency of DGK leads to modest growth retardation, reduced adiposity, and protection against peripheral insulin resistance in HFD-fed mice. DGK ablation increases DAG content in peripheral tissues controlling glucose and energy homeostasis. Moreover, DGK plays a role in skeletal muscle differentiation, likely due to a fundamental role during development. Thus, DGK plays a role in the control of growth and metabolic processes and further highlights specialized functions of DGK isoforms in glucose and energy homeostasis and type 2 diabetes pathophysiology.